New - Biotechnology (Class XII)
New - Biotechnology (Class XII)
BIOTECHNOLOGY
CLASS - XII
CBSE
Price : Rs.
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DESIGN & LAYOUT : Multi Graphics, 5745/81, Reghar Pura, Karol Bagh,
New Delhi - 110005.
PRINTED BY
First Edition
Central Board of Secondary Education. Delhi
All rights reserved. No part of this publication may be reproduced in any form or by any means, electronic,
mechanical, photocopying, recording or otherwise without prior permission of the publisher.
First Edition: March 2003
ADVISORY COMMITTEE
c
Mr Ashok Ganguly, Chairman, CBSE
c
Mr G. Balasubramanian, Director (Academic), CBSE
c
Prof. K. Kannan, Convenor, CBSE Committee of Courses in Biotechnology
EDITORS
c
Prof. K. Kannan, Dean, School of Biotechnology, Guru Gobind Singh lndraprastha University, Delhi
c
Dr K. Nirmala, Reader, Department of Biochemistry, Daulat Ram College, University of Delhi,
Delhi
CONTRIBUTORS
c
Prof. K. Kannan, Dean, School of Biotechnology, Guru Gobind Singh Indraprastha University, Delhi
(Convenor, CBSE Committee of Courses in Biotechnology)
c
Dr Vijay Kumar, Senior Research Scientist, International Centre of Genetic Engineering and
Biotechnology, Aruna Asaf Ali Marg, New Delhi
c
Dr V.L. Kumar, Additional Professor, Department of Pharmacology, All India Institute of Medical
Science, New Delhi.
c
Dr M.V. Rajam, Reader, Department of Genetics, University of Delhi, South Campus, New Delhi
c
Dr Amulya K Panda, Scientist, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi
c
Dr J.S. Virdi, Reader, Department of Microbiology, University of Delhi, South Campus, New Delhi
c
Dr S. Ramachandran, Scientist, Institute of Genomics & Integrative Biology, Mall Road, Delhi
c
Dr Rajesh S. Gokhale, Scientist, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi
c
Dr Pushkar Sharma, Scientist, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi
c
Dr K.K. Aggarwal, Lecturer, School of Biotechnology. Guru Gobind Singh Indraprastha University,
Delhi
c
Dr Ashwani Pareek, Lecturer, School of Biotechnology, Guru Gobind Singh Indraprastha University,
Delhi
c
Dr Nimisha Sharma, Lecturer, School of Biotechnology, Guru Gobind Singh Indraprastha
University, Delhi
c
Dr Promila Gupta, Lecturer, School of Biotechnology, Guru Gobind Singh Indraprastha University,
Delhi
c
Mr Suresh Kumar, Lecturer, School of Biotechnology, Guru Gobind Singh Indraprastha University,
Delhi
DESIGN, LAYOUT & ILLUSTRATIONS :
Dr Ashwani Pareek, Mr. Ramesh Chand Sharma, Ms. Tannistha Nandi, School of Biotechnology, Guru Gobind
Singh Indraprastha University, Delhi
PROJECT CO-ORDINATOR :
Mr R.P. Sharma, Education Officer (Science), CBSE
PUBLISHED BY:
Mr B.R. Sharma, Secretary, Central Board of Secondary Education, 'Shiksha Kendra', 2, Community Centre,
Preet Vihar, Delhi - 110 092
Second Edition
Central Board of Secondary Education, Delhi
All rights reserved. No part of this publication may be reproduced in any form or by any means,
electronic, mechanical, photocopying, recording or otherwise without prior permission of the
publisher.
Second Edition: June 2011
ADVISORY COMMITTEE
c
Sh. Vineet Joshi, Chairman, CBSE
c
Ms. Chitralekha Gurumurthy, Director (Academic), CBSE
c
Prof. M. V. Rajam, Convenor, CBSE Committee of Courses in Biotechnology
EDITOR
c
Prof. M. V. Rajam, University of Delhi, South Campus, New Delhi
CONTRIBUTORS
c
Prof. M. V. Rajam
Department of Genetics, University of Delhi, South Campus, New Delhi
c
Prof. J. S. Virdi
Department of Microbiology, University of Delhi, South Campus, New Delhi
c
Dr K. Nirmala
Associate Professor, Department of Biochemistry, Daulat Ram College, University of
Delhi, Delhi
c
Dr Vijay Kumar
Staff Scientist, International Centre of Genetic Engineering and Biotechnology, Aruna
Asaf Ali Marg, New Delhi
c
Dr S. Ramachandran
Scientist E2, Institute of Genomics & Integrative Biology, Mall Road, Delhi
COVER DESIGN & LAYOUT
c
Prof. M. V. Rajam
Department of Genetics, University of Delhi, South Campus, New Delhi
PROJECT CO-ORDINATORS
c
Mr. P.V. Sai Ranga Rao and Dr. Rashmi Sethi, Education Officers (Science), CBSE
PUBLISHED BY
Secretary, Central Board of Secondary Education, 'Shiksha Kendra', 2, Community Centre, Preet
Vihar, Delhi - 110 092
Ashok Ganguly
Chairman, CBSE
VINEET JOSHI
CHAIRMAN
Any textbook requires meticulous and painstaking efforts by all its contributors. We are grateful
to all the contributors who have cheerfully revised and re-revised their sections to keep the
subject level interesting and readable at the class XII level. In addition, several other have made
contributions in reading and recommending changes wherever necessary and these include
students from the School of Biotechnology, GGSIPU. Special thanks are due to Anjana, Neeraj,
Bhavana, Shruti, Ritu, Nitika, Amit, Yasha, Archana, Aditya and Deepak Grover. We would like to
acknowledge the assistance of Mr Sanjeev Kumar and Mr Amreesh Kumar in preparation of the
book.
The Convener and the Resource team are ever so grateful to Mr. Ashok Ganguly, Chairman CBSE,
Mr. G Balalsubramanian, Director Academic and Mr. R P Sharma, Education Officer (Science), CBSE
whose complete faith in the team has had an undoubted influence on much of the content.
Without the help of these people, the book could not have seen the light of the day. I can only
hope that they will all look upon the results of the influence and endeavor with pleasure.
Wherever possible, credit has been given and if I have failed to acknowledge the source of an idea
or a technique, it is because the source for some reason is unknown to me. In the rapidly
expanding world of biotechnology, it is often impossible to know where the idea really originated.
We hope that all the budding biotechnologists will fulfill our dream and convert India into a
sustainable developed nation.
Prof. K. Kannan
Convenor
CBSE Committee of Courses in Biotechnology
Prof. M. V. Rajam
Convenor
CBSE Committee of Courses in Biotechnology
Subs, by the Constitution (Forty-Second Amendment) Act. 1976, sec. 2, for "Sovereign Democratic Republic (w.e.f. 3.1.1977)
Subs, by the Constitution (Forty-Second Amendment) Act. 1976, sec. 2, for "unity of the Nation (w.e.f. 3.1.1977)
(d) to defend the country and render national service when called upon to do so;
(e) To promote harmony and the spirit of common brotherhood amongst all the people of India
transcending religious, linguistic and regional or sectional diversities; to renounce practices
derogatory to the dignity of women;
(f)
(g) to protect and improve the natural environment including forests, lakes, rivers, wild life and to have
compassion for living creatures;
(h) to develop the scientific temper, humanism and the spirit of inquiry and reform;
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constantly rises to higher levels of endeavour and achievement.
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Contents
UNIT 5 : PROTEIN AND GENE MANIPULATION
CHAPTER 5.1 : RECOMBINANT DNA TECHNOLOGY
5.1.1.
Introduction
5.1.2.
5.1.3.
Making rDNA
13
5.1.4.
14
5.1.5.
Identification of Recombinants
15
5.1.6.
17
5.1.7.
Hybridization Techniques
19
5.1.8.
DNA Library
21
5.1.9.
DNA Sequencing
22
5.1.10.
Site-directed Mutagenesis
26
29
5.2.2.
30
5.2.3.
34
5.2.4.
Purification of Proteins
38
5.2.5.
Characterization of Proteins
44
5.2.6.
47
5.2.7.
51
Introduction
57
5.3.2.
60
5.3.3.
61
5.3.4.
62
5.3.5.
Functional Genomics
64
5.3.6.
Proteomics
69
5.3.7.
History of Bioinformatics
72
5.3.8.
74
5.3.9.
Information Sources
78
5.3.10.
80
Contents
UNIT 6 : CELL CULTURE AND GENETIC MANIPULATION
CHAPTER 6.1 : MICROBIAL CELL CULTURE AND ITS APPLICATION
6.1.1.
Introduction
85
6.1.2.
85
6.1.3.
92
6.1.4.
97
6.1.5.
99
6.1.6.
101
6.1.7.
104
6.1.8.
106
Introduction
109
6.2.2.
109
6.2.3.
114
6.2.4.
119
6.2.5.
122
6.2.6.
130
Introduction
135
6.3.2
136
6.3.3
142
6.3.4
143
6.3.5
144
6.3.6
145
6.3.7
148
6.3.8
Tissue Engineering
152
5
PROTEIN AND
UNIT
GENE MANIPULATION
CHAPTER
RECOMBINANT
DNA TECHNOLOGY
1
5.1.1. Introduction
Every day the newspapers tell you about the remarkable feats of science and technology in
helping man combat disease and improve his environment. Many of these involve the use of a
powerful technique called gene cloning or Recombinant DNA Technology (RDT). Using such
technology bacteria in the past were engineered to produce human insulin a hormone which
fights diabetes, yeast cells were made to produce Hepatitis B vaccine, plants such as cotton were
made insect resistant (Bt-cotton) and even as you read this chapter projects are on to engineer
bacteria to cleanup environmental waste such as polythene. Have you not wondered how
Scientists are able to achieve all this? The present chapter will introduce you to the main
techniques used in gene cloning along with some important applications for you to understand
and marvel at the simplicity and power of this area of biotechnology.
The basic steps involved in RDT are illustrated schematically below Fig. 1 :
DNA fragment
Closing vector (plasmid)
Cut with enzyme
Ligate together
Insert containing gene of interest
Recombinant plasmid
Introduce plasmid into E.coli host cell (transformation)
chromosome
Host cell division
UNIT
Isolation of a DNA fragment containing a gene of interest that needs to be cloned (called as
insert).
2.
Generation of a recombinant DNA (rDNA) molecule by insertion of the DNA fragment into a
carrier DNA molecule called vector (e.g. plasmid) that can self replicate within a host cell.
3.
Transfer of the rDNA into an E. coli host cell (process called transformation).
4.
Selection of only those host cells carrying the rDNA and allowing them to multiply thereby
multiplying the rDNA molecules.
The whole process thus can generate either a large amount of rDNA (gene cloning) or a large
amount of protein expressed by the insert. The first rDNA molecules to be generated using these
procedures were established by the combined efforts in 1973 by the molecular biologists Paul
Berg, Herbert Boyer, Annie Chang and Stanley Cohen.
A restriction enzyme that selectively recognises a specific DNA sequence and digests any
DNA fragment containing that sequence. The term restriction is derived from the ability of
these enzymes to restrict the propagation of foreign DNA (e.g. Viral/phage DNA) in a
bacterium.
2.
A modification enzyme that adds a methyl group to one or two bases within the sequence
recognised by the enzyme. Once a base is modified by methylation, the sequence cannot be
digested. It is thus obvious that the Restriction-Modification enzyme system within a given
bacterium protects its DNA from digestion by methylation but can digest foreign DNA which
is not protected by similar methylation.
Different species of bacteria contain their own sets of restriction endonucleases and
corresponding methylases. Three main classes of restriction endonucleases- type I, type II and
type III are present, of which, only type II restriction enzymes are used in rDNA technology as they
UNIT
recognise and cut DNA within a specifc sequence typically consisting of 4-8 bp. This sequence is
referred to as a restriction site and is generally palindromic, which means that the sequence in
both DNA strands at this site read same 5' to 3' direction. Type II restriction enzymes are named
after the bacterial species they have been isolated from. For example a commonly used
restriction enzyme EcoRI isolated from the bacterial species E. coli is named so with the first
three italicised alphabets referring to the genus (E) and species (co), the capital R referring to the
strain (RY 13) and the number designated with the roman numeral (I) indicating that it was the
first enzyme to be isolated from this strain of bacteria. Restriction enzymes were first discovered
and studied by the molecular biologists W. Arber, H. Smith and D. Nathans for which they were
awarded the Nobel Prize in 1978. Today more than a thousand restriction enzymes are available
for genetic engineers to use. Some commonly used restriction enzymes (type II) along with their
source and restriction- modification sequences have been listed in Table1 below.
Table 1. Type II restriction enzymes, their sources, recognition and cleavage sites.
Restriction enzyme
Microbial source
Recognition sequence
Alu I
Arthrobacter luteus
5'A-G-C-T 3
3'T-C-G-A 5
BamHI
Bacillus amyloliquefaciens
5'G-G-A-T-C-C 3'
3'C-C-T-A-G-G 5'
EcoRI
Escherichia coli
5'G-A-A-T-T-C 3'
3'C-T-T-A-A-G 5'
EcoRII
Escherichia coli
5'C-C-T-G-G 3'
3'G-G-A-C-C 5
HaeIII
Haemophilus aegyptus
5'G-G-C-C 3
3'C-C-G-G 5
HindIII
Haemophilus influenza
5'A-A-G-C-T-T 3
3'T-T-C-G-A-A 5
PstI
Providencia stuartii
5'C-T-G-C-A-G 3
3'G-A-C-G-T-C 5
SalI
UNIT
Streptomyces albus
5'G-T-C-G-A-C 3
3'C-A-G-C-T-G 5'
The exact kind of cleavage produced by a restriction enzyme is important in the design of a gene
cloning experiment. Some cleave both strands of DNA through the centre resulting in a blunt or
flush end. These are also known as symmetrical cuts. From Table I it is obvious that the enzyme
AluI cuts symmetrically.
5'-A-G-C-T-3'
5'-A-G
3'-T-C-G-A-5'
C-T-3'
3'-T-C
G-A-5'
However EcoRI cuts in a way producing protruding and recessed ends known as sticky or
cohesive ends because these ends can base pair and stick the DNA molecule back together again.
Such cuts are termed staggered.
5'-G-A-A-T-T-C-3'
5'-G-3'
3'-C-T-T-A-A-G-5'
3'-C-T-T-A-A-5'
5'-A-A-T-T-C-3'
3'-G-5'
Note that EcoRI generates 5' overhangs at the cut site (and 3' recessed ends). Hence if two
different DNA fragments containing EcoRI recognition sites are cleaved and mixed the sticky ends
can bind and generate a hybrid or recombinant DNA (Fig.2)
Fragment B
Fragment A
Cut with EcoRI
Sticky end
UNIT
Individual A
Individual B
Isolate DNA
Individual A
UNIT
Individual B
Individuals except identical twins vary in their RFLP pattern as indicated schematically in
the agarose gel electrophoresis. Hence the term DNA fingerprint is used and this is the basis of a
major technique used in forensic science to identify and relate individuals.
1.
2.
It should incorporate a selectable marker, a gene whose product can identify the host
cells containing the vector. Selectable markers include genes conferring antibiotic
resistance, enzymes such as
-galactosidase which can turn substrates blue in the
vicinity of the host cell colony and gene expressing Green Fluorescent Protein (GFP)
which cause host cells containing the vector to fluoresce when viewed under UV light.
UNIT
3.
The vector must also have one unique restriction enzyme recognition site which can be
used for cutting and introducing an insert. Most of the commonly used cloning vectors
have more than one restriction site, they contain a Multiple Cloning Site (MCS) or
polylinker. The MCS provides flexibility in the choice and use of restriction enzymes.
4.
Another desirable feature of a cloning vector is that it should be small in size thereby
facilitating entry/transfer into a host cell.
A number of vectors have been developed incorporating these features but only
plasmids and bacteriophage based vectors will be discussed.
Plasmids
Plasmids are extrachromosomal, self-replicating, usually circular, double-stranded DNA
molecules found naturally in many bacteria and also in some yeasts. Although plasmids are not
essential for normal cell growth and division, they often confer useful properties to the host such
as resistance to antibiotics that can be a selective advantage under certain conditions. The
plasmid molecules can be present as 1 or 2 copies or in multiple copies (500-700) inside the host
organism. These naturally occurring plasmids have been modified to serve as vectors in the
laboratory and are by far the most widely used, versatile and easily manipulated vectors.
One of the earliest plasmid vectors to be constructed was pBR322 (Fig. 4a). This plasmid contains
two different antibiotic resistance genes and recognition sites for several restriction enzymes. A
popular series of plasmid cloning vectors is the pUC family (Fig. 4b). These vectors contain a
region of the lacZ gene that codes for the enzyme
-galactosidase. This region also contains a
polylinker and thus insertion of a foreign DNA into any of the restriction sites will result in an
altered non-functional enzyme. During screening of recombinant plasmid containing host cells
the absence of
-galactosidase activity is indicative of plasmids containing the insert.
The plasmid vectors described above can replicate only in E. coli. Many of the vectors used in
eucaryotic cells are constructed such that they can exist both in the eukaryotic cell and E. coli.
Such vectors are known as shuttle vectors. These vectors contain two types of origin of replication
and selectable marker genes, one set which functions in the eukaryotic cells (e.g. yeast) and
another in E. coli. An example of a shuttle vector is the yeast plasmid Yep (Fig. 4c). In the case of
plants, a naturally occurring plasmid of the bacterium Agrobacterium tumefaciens called Ti
plasmid has been suitably modified to function as a vector.
UNIT
EcoRI
b.pUC 19
a. pBR322
c. YEp
E.Coli ori
Yeast ori
AmpR
LEU2 gene
-gal
TetR
10
UNIT
Bacteriophage lambda has a double stranded, linear DNA genome containing 48,514 bp, in which
12 bases on each end are unpaired but complementary. These ends therefore are sticky and are
called cohesive or cos sites and are important for packaging DNA into phage heads. An important
feature of the lambda genome is that a large fragment in the central region of its genome is not
essential for lytic infection of E. coli cells. Therefore, vectors have been designed such that this
region can be replaced by foreign insert DNA. These phage based vectors allow cloning of DNA
fragments up to 23 kb in size.
M13 is a filamentous phage which infects E. coli having a pilus (protrusion) which is selectively
present in cells containing a F plasmid (called F+ cells). The genome of the M13 phage is a single
stranded, circular DNA of 6407 bp. Foreign DNA can be inserted into it without disrupting any of
the essential genes. In the life cycle of the phage following infection of the host E.coli cell the
single stranded DNA is converted to a double-stranded molecule which is referred to as the
Replicative Form (RF). The RF replicates until there are about 100 copies in the cell. At this point
DNA replication becomes asymmetric and single stranded copies of the genome are produced and
extruded from the cell packaged with protein as M13 particles (Fig. 5). The major advantages of
developing vectors based on M13 are that its genome is less than 10 kb in size; the RF can be
purified and manipulated exactly like a plasmid. In addition, genes cloned into M13 based vectors
can be obtained in the form of single stranded DNA. Single stranded forms of cloned DNA are
useful for use in various techniques including DNA sequencing and site-directed mutagenesis,
techniques which will be discussed in a latter section.
M13 phase
(single strand)
RF
(double strand)
Assymmetric synthesis
UNIT
11
Cosmids
Cosmids have been constructed by combining certain features of plasmid and the 'cos' sites of
phage lambda. The simplest cosmid vector contains a plasmid, origin of replication, a selectable
marker, suitable restriction enzyme sites and the lambda cos site. Cosmids can be used to clone
DNA fragments up to 45 kbp in size.
YAC vectors
YACs or Yeast Artificial Chromosomes are used as vectors to clone DNA fragments of more than 1
Mb in size. Therefore, they are useful in cloning larger DNA fragments as required in mapping
genomes such as in the Human Genome Project. These vectors contain a teleomeric sequence,
the centromere and an autonomously replicating sequence, features required to replicate linear
chromosomes in yeast cells. These vectors also contain suitable restriction sites to clone foreign
DNA as well as genes to be used as selectable markers.
BAC vectors
BACs or Bacterial Artificial Chromosomes are vectors based on the natural, extrachromosomal
plasmid from E. coli - the fertility or F plasmid. A BAC vector contains genes for replication and
maintenance of the F plasmid, a selectable marker and cloning sites. These vectors can
accommodate inserts up to 500 kb and are used in genome sequencing projects. Table 2 lists the
common cloning vectors with the size of insert that can be cloned into them.
Table 2. Common cloning vectors.
Vector Type
Plasmid
0.5-8
Bacteriophage lambda
9-23
Cosmid
30-40
BAC
50-500
YAC
250-1000
12
UNIT
to adsorb to cells, introduce their DNA and replicate, have made them ideal vehicles to transfer
foreign DNA into eukaryotic cells in culture. A vector based on Simian Virus 40 (SV40) was used in
the first cloning experiment involving mammalian cells. A number of vectors based on other type
of viruses like Adenoviruses and Papillomavirus have been used to clone genes in mammals. At
present, retroviral vectors are popular for cloning genes in mammalian cells. In case of plants,
viruses like Cauliflower Mosaic Virus, Tobacco Mosaic Virus and Gemini viruses have been used
with limited success.
Host Cells
The tools described in the previous sections will result in the generation of recombinant DNA
molecules in the laboratory. Eventually the propagation of these DNA molecules must occur inside
a living system or host. Many types of host cells including E. coli, yeast, animal and plant cells are
available for gene cloning and the type of host cell used depends on the aim of the cloning
experiment. E. coli has become the most widely used organism in rDNA technology because its
genetic make-up has been intensively studied, it is easy to handle and grow, can accept a range of
vectors and has been extensively studied for safety. Another major advantage of using E. coli as
host cells is that under optimal conditions the cells divide every 20 minutes making it possible to
clone large amounts of foreign DNA and if the appropriate signals are incorporated into the vector
large amounts of recombinant proteins are available for therapeutics and other uses.
For the expression of eukaryotic proteins, eukaryotic cells are often preferred because to be
functionally active, proteins require proper folding and post translational modifications such as
glycosylation which is not possible in prokaryotic (E. coli) cells. Even cloned eukaryotic genes
containing introns cannot be processed in E. coli thereby necessitating the use of only eukaryotic
host cells. Yeast cells have been used extensively for functional expression of eukaryotic genes
because of several features. Yeasts are the simplest eukaryotic organisms (unicellular) and like
E. coli have been extensively characterised genetically, easy to grow and manipulate and large
amounts of cloned genes or recombinant proteins can be obtained from yeast cultures grown in
fermentors (large culture vessels). Plant and animal cells may also be used as hosts in rDNA
experiments and cells can be grown in tissue culture or can be induced and manipulated to form
whole organisms (creation of transgenic animals and plants).
UNIT
13
Ori
Foreign DNA
Plasmid vector
Cut DNA fragment and plasmid vector
Antibiotic resistance gene
with restriction enzyme (EcoRI)
Sticky ends
14
UNIT
Transformation: In rDNA technology, the most common method to introduce rDNA into living cells
is called transformation. In this procedure, bacterial cells take up DNA from the surrounding
environment. Many host cell organisms such as E. coli, yeast and mammalian cells do not readily
take up foreign DNA and have to be chemically treated to become competent to do so. In 1970,
Mandel and Higa found that E. coli cells become markedly competent to take up external DNA
when suspended briefly in cold calcium chloride solution.
Transfection: Another method to transfer rDNA into host cells involves mixing the foreign DNA
with charged substances like calcium phosphate, cationic liposomes or DEAE dextran and
overlaying on recipient host cells. Host cells take up the DNA in a process called transfection.
Electroporation: An electric current is used to create transient microscopic pores in the
recipient host cell membrane allowing rDNA to enter.
Microinjection: Exogenous DNA can also be introduced directly into animal and plant cells
without the use of eukaryotic vectors. In the procedure of microinjection, foreign DNA is directly
injected into recipient cells using a fine microsyringe under a phase contrast microscope to aid
vision.
Biolistics: A remarkable method that has been developed to introduce foreign DNA into mainly
plant cells is by using a gene or particle gun. Microscopic particles of gold or tungsten are coated
with the DNA of interest and bombarded onto cells with a device much like a particle gun. Hence
the term biolistics is used.
Another method of introducing foreign genes is by the natural genetic engineer Agrobacterium
tumefaciens. This method and its principle will be covered in the chapter of plant cell culture in
Unit VI.
UNIT
15
Fig. 7. Replica plating (note only colonies marked yellow have insert).
16
UNIT
Another powerful method of screening for the presence of recombinant plasmids is referred to as
blue- white selection. This method is based upon the insertional inactivation of the lac Z gene
present on the vector (e.g. pUC 19). This gene expresses the enzyme
-galactosidase whose
activity can cleave a colourless substrate called X-Gal into a blue coloured product. If the lac Z
gene is inactivated due to the presence of the insert then the enzyme is not expressed. Hence if
after a transformation experiment the E. coli host cells are plated on an ampicillin and X-Gal
containing solid media plate then colonies which appear blue are those which have transformed
cells (antibiotic resistant) but do not have the insert (express active enzyme). Colonies which
appear white are both ampicillin resistant and have the insert recombinant DNA and thus are the
cells to be used for future experiments.
The above described methods are used for selection of E. coli recombinants. There are several
methods used for other host cell recombinants but the principles remain the same. Furthermore
techniques for actually detecting recombinant proteins from colonies also have to be used where
relevant. Where amplification of the insert DNA is the primary objective, plasmids are isolated
from the host cells after growing the latter in large amounts and using the same restriction
enzyme the insert is cut from the plasmid and recovered after electrophoresis.
2.
Primers which are oligonucleotides, usually 10-18 nucleotides long that hybridise to
the target DNA region one to each strand of the DNA. Two primers of such a sequence
are required so that they can hybridise as indicated in the Fig. 8.
3.
DNA polymerase which is stable at temperatures above 80oC . Taq polymerase which
has been isolated from a thermostable bacterial species is used.
4.
UNIT
17
5
3
Denaturation step
Above 800C
Annealing step
Extension step
Primers
500-600C
Above 700C
18
UNIT
amplified from the small amounts present at the crime site, for DNA fingerprinting analysis. In
recent years, PCR has also found use in detecting specific microorganisms from environmental
samples of soil, sediments and water. It is interesting that archaeologists are using combinations
of PCR and fingerprinting analysis to relate and establish ancient Egyptian dynasties from samples
obtained from mummies.
Fluorescent probe
UNIT
19
Digest
Fragments
Genomic DNA
Weight
Agarose gel pattern
Nitrocellulose
} Tissue paper
Agarose gel
Support
wick
through with buffer
Nitrocellulose blot
Hybridize with probe
20
UNIT
UNIT
21
As shown in Fig. 11, to prepare a genomic library, the total genomic DNA is isolated from a tissue
or organism and then fragmented using a restriction enzyme such as EcoRI. An appropriate vector
such as pBR322 (plasmid based) is also digested with the same enzyme and is then treated with
the enzyme alkaline phosphatase which as indicated elsewhere removes the 5'phosphate group to
prevent the plasmid from self ligation. The DNA fragments and cut vector are mixed and then
treated with the enzyme DNA ligase. Each vector molecule will contain a different fragment of
DNA and these are introduced into E. coli host cells by a technique called transformation. More
details about this technique will be discussed in the following section.
Although a genomic library represents the entire genome of an organism, it may not be useful in
case of eukaryotic organisms. Genomic DNA of eucaryotes contains non-coding DNA like introns,
control regions and repetitive sequences unrecognised by prokaryotic systems such as in E. coli.
Therefore another library called cDNA library is preferred especially if the aim of the cloning
experiment is to express eukaryotic proteins in an E. coli host. This library has two major
advantages over a genomic library. Firstly, it represents only those genes that are being expressed
by a particular cell or under specific conditions. Eucaryotes, which include multicellular
organisms such as animals and plants have differentiated cells which means that different cells
e.g. liver and brain cells express some common proteins and also other proteins which are not
common. Secondly, since the source material in constructing such libraries is mRNA, these
molecules lack introns and hence would represent only the coding sequences of the genome.
However mRNA molecules are highly unstable as they are easily degraded by RNAses and hence
these molecules are faithfully copied into the more stable DNA (now called cDNA) before cloning.
The construction of a cDNA library begins with the isolation of mRNA from a given cell type or
tissue which are copied into cDNA using a special enzyme called reverse transcriptase. The
procedure results in double-stranded cDNA which can be incorporated into vectors such as
pBR322.
The dideoxynucleotide chain termination method invented by Fred Sanger (the same
scientist who invented a protein sequencing ) and Andrew Coulson
2.
Chemical degradation method invented by Walter Gilbert and Allan Maxam (also
called the Maxam and Gilbert method).
Of the two methods the first method is more popularily used and hence this technique
will be discussed.
22
UNIT
A new strand cannot be initiated; only primers can be extended using the single strand
DNA template as a guide.
Pi
Pi
Pi
3
OH
2 deoxynucleotide triphosphate
Base (A/T/C/G)
Pi
Pi
Pi
3
H
2
H
UNIT
23
AACGAGATGCCTT
Primer
DNA polymerase
AACGAGATGCCTT
CTCTTA
Template strand
Typically the sequencing technique is carried out in four test tubes. Each test tube carries the
following common reagents, single stranded DNA template, deoxynucleotide triphosphates,
primer and DNA polymerase. In addition to it a small amount of the four dideoxynucleotide
triphosphates i.e ddATP, ddCTP, ddGTP and ddTTP is added separately into the four test tubes.
The purpose of adding only a small amount of the dideoxy derivative is that only one dideoxy
derivative is incorporated into each extending chain causing termination. Hence in a test tube
containing ddATP all chains will end at ddA but at different positions of T present in the template
(Fig. 14). The same is applicable to the other test tubes containing ddCTP, ddGTP and ddTTP. It is
to be noted that the dideoxynucleotide is added wherever the complementary base is present on
the template.
24
UNIT
AACCGGTACTGGCAT
TTGGCCA
All strands end in ddA
TTGGCCATGA
TTGGCCATGACCGTA
Smaller strands
ddT
ddG
ddC
Sequence readout
G 3
C 5
A 5
T 3
+
New strand
UNIT
Original strand
Fig. 15. Reading a sequencing gel pattern (lines drawn for clarity in reading).
25
Nowadays DNA sequencing technologies have become automated. To avoid using radioisotopes
and their consequent danger, dideoxynucleotides are conjugated with florescent molecules
which on excitation give a different colour each. Hence each band on the gel (read from anode to
cathode) indicates the particular base as its terminal dideoxy nucleotide fluoresces with a given
colour. This avoids the use of a four lane gel, a single lane gel electrophoresis is instead conducted
and the gels are then laser scanned and the data fed into a computer. The computer is
programmed to display the gel scan and the base readout as shown in Fig. 16.
Fluorescence
detector
Migration of DNA
fragments
Laser
Sample/
Buffer
Buffer
+
Fig. 16. Single Tube Sequencing experiment.
26
UNIT
molecule is then introduced into bacterial cells by transformation. Subsequent replication inside
bacterial cells will produce either wild type or mutant gene containing plasmids. If appropriate
expression signals are present altered protein can be expressed and studied. In the unit on Protein
engineering a site-directed mutagenesis experiment has been described wherein a stable form
of the proteolytic enzyme subtilisin has been generated which is used in laundry detergent.
Oligo with mutation (s)
In vitro synthesis of RF
mutant
Wild type
Wild type
mutant
Replication in bacteria
RF
Review Questions
1.
UNIT
Restriction site
Mutation
27
2.
What are restriction enzymes and why are they so important in rDNA technology?
3.
4.
5.
What does PCR stand for? Name the different steps in a PCR reaction.
6.
7.
What are the disadvantages of using E. coli for production of eukaryotic proteins?
8.
Distinguish between:
i) Blunt ends vs sticky ends
ii) YAC vs BAC
iii) Genomic library vs cDNA library
iv) Microinjection vs Electroporation
9.
Write a short note on RFLP and indicate one of its important applications.
10.
Why are ddNTPs used in sequencing? Briefly indicate the principle of DNA sequencing using
these.
11.
References
1. Molecular Biotechnology by S.B. Primrose, Panima Publishing Corporation, New Delhi (1999),
2nd Edition.
2. Molecular Biotechnology by Glick and Pasterneck, Panima Publishing Corporation, New Delhi
(1999), 2nd Edition.
3. An Introduction to Molecular Biotechnology Edited by Michael Wink, Wiley-VCH Verlag GmbH &
Co. KGaA, Weinheim, Germany (2006).
4. Principles of Gene Manipulation and Genomics by S. B. Primrose and R. M. Twyman, Blackwell
Publishing, Carlton, Victoria, Australia (2006), 7th Edition.
5. From Genes to Clones by Ernst-L. Winnacker, Panima Publishing Corporation, New Delhi
(2003).
6. Gene Cloning and DNA Analysis: An Introduction by T. A. Brown, Blackwell Sciences Ltd., Oxford
(2001), 4th Edition.
28
UNIT
CHAPTER
PROTEIN STRUCTURE
AND ENGINEERING
Collagen
UNIT
29
these functions and why are some brain related diseases like Alzeimers occurring, in which
certain proteins show abnormal structure and behaviour?
A number of human diseases are due to the deficiency or abnormal structure of proteins. The lack
of a particular subunit, alpha or beta of the oxygen carrying protein haemoglobin results in
Thalassaemia, a devastating disease in which an infant cannot grow without repeated
transfusions. If the beta chain is present but with a substituent in one of the amino acid residues
another debilitating condition called Sickle Cell Anaemia results which is endemic to certain parts
of Africa. The absence of an enzyme- Adenosine deaminase results in the birth of a severely
immunocompromised baby who cannot last infancy (SCID). More recently it has been discovered
that certain "rogue proteins" whose structure has been altered can result in diseases such as the
Mad cow disease wherein the disease itself appears to be propagated by infectious proteins called
"prions". Clearly proteins need to be understood in detailed terms.
The completion of the Human Genome Sequence has revealed about 35,000 genes. However the
actual number of proteins encoded by these genes may be many more due to posttranscriptional
modifications. Different cells have specialised proteins for their unique functions in addition to
the housekeeping proteins required for metabolism and generation of ATP. Sometimes these
proteins are secreted to the outside like the proteolytic enzymes from the pancreas or hormones
from ductless glands like the pituitary. We are yet to identify all the proteins required for a body
to function and this presents a challenge to the future biotechnologists. One of the outcomes is
the merging field of protein structure and function- proteomics. This chapter will enable you to
understand various features of the area of proteomics- 3-D structure, functions and applications
of protein products, some generated by biotechnological processes.
30
UNIT
One of the major breakthroughs in protein sequence determination was achieved in the middle of
the last century by Dr. Frederick Sanger who developed the first sequencing reagent FDNB (fluoro
dinitro benzene) and a general strategy for sequencing. By using these methods he was able to
sequence the important hormone insulin which is required by diabetics and more importantly he
demonstrated for the first time that proteins were linear polymers of amino acids. For this work
he was awarded the Nobel Prize and it will be interesting for you to know that several years later
he was awarded a second Nobel Prize for developing a sequencing technique for DNA which has
been described in the Recombinant DNA technology chapter previously. Another protein chemist,
Pehr Edman in 1950 developed another sequencing reagent and procedure which is used in
modern day sequenators as the procedure has been automated. These methods have been
th
discussed in the XI class textbook of Biotechnology in Unit II, Chapter 2. Notably using the
sequence of insulin established by Sanger a biotechnology company called Eli Lilli was able to
develop recombinant human insulin which is the major source for insulin administration to
diabetics worldwide.
With the availability of pure proteins, scientists like Linus Pauling, G.N.Ramachandran, Max
Perutz and John Kendrew to name a few started developing techniques to study the 3-D shapes of
proteins using high resolution X-rays. They laid the foundation for deducing protein structure by
enunciating the basic rules which govern protein folding and the forces which cause the folding
and stabilise them. Hence from these studies the concepts of planarity of the peptide bond,
secondary structures such as alpha helix and beta pleats were developed. These concepts were
introduced in the Class XI Biotechnology textbook.
Let us reiterate some important points regarding protein structure from the Class XI
Biotechnology textbook. Protein structure has been divided into four hierarchial levels to
understand their organisation:
The linear order or sequence of covalently linked amino acid sequence is defined as primary
structure. Depending on the nature and arrangement of the amino acids present different parts
of the polypeptide chain form secondary structures like alpha helices and beta pleats. The
tertiary structure organisation of these secondary structural elements occurs when these get
compacted with each other to form compact spherical or globular units which are also
thermodynamically stable conformations of these molecules in aqueous solutions (note
cytoplasm is mainly water). In compaction several non-covalent interactions occur between the
amino acid side chains. The quarternary structure is the association of two or more independent
proteins/polypeptides via non-covalent forces to give a multimeric protein (Fig. 2). The
individual peptide units of this protein are referred to as subunits and they may be identical or
different from one another.
The dominant forces which cause linear protein chains to undergo folding in space lies to a large
extent in the chemistry of the amino acid residues they contain. Amino acids are broadly divided
UNIT
31
into three main groups- polar or hydrophilic (eg. serine, glutamine), charged (eg. aspartate,
arginine) and hydrophobic (eg. tryptophan, valine). Hence based on these features amino acid
side chains can interact in space by a variety of non-covalent forces which is the basis of forming
and stabilising protein structures in space. Let us examine some of the major non-covalent forces
found in proteins.
Non-covalent bonds
The non-covalent interactions involved in organising the structure of protein molecules can be
broadly divided into four categories:
Ionic bonds
Hydrogen bonds
Hydrophobic interactions
Ionic bonds
These involve interactions between the oppositely charged groups of a molecule. For example
the positively charged amino acid side chains of lysine and arginine can form salt bridges with the
negatively charged side chains of aspartate and glutamate. These ionic interactions are also
known as salt bridges because these are dominant bonds found in salts like sodium chloride
wherein the positively charged sodium ion interacts with the negatively charged chloride ion.
However, although ionic bonds have similar strengths to covalent bonds in vacuo, the bond
strength of ionic bonds is vastly reduced in water due to the insulating qualities (dielectric
strength) of water. Ionic bonds are highly sensitive to pH and salt concentration.
32
UNIT
Hydrogen bonds
Hydrogen bonds are formed by "sharing" of a
hydrogen atom between two electronegative atoms
such as Nitrogen and Oxygen. In this case strongly
polarised bonds between hydrogen and a small, very
electronegative atom (N,O or F) allow a strong
dipole-dipole bond to be formed with another small
very electronegative element (N, O or F, Fig. 3).
Importantly, the very small sizes of these elements
also allow them to approach each other so closely
that a partial covalent bond is also formed
(e.g.O-H---N). It is to be noted that the partial
covalent character means that these bonds (Hbonds) are directional and strongest when the nuclei
of all three involved atoms are in a linear arrangement.
water
Hydrophobic interactions
Hydrophobic interactions can be best explained by taking an example of oil in water. The oil tends
to separate out fairly quickly, not because the oil molecules "want to get together", but because
the water forces them out. The hydrophobic interaction is thus a manifestation of hydrogen
bonding network in water. In water, each molecule is potentially bonded to four other molecules
through H-bonds (Fig. 3).
If a non-polar molecule, which cannot participate in hydrogen bonding, or in electrostatic
interactions with water molecules, is added into water , a number of hydrogen bonds will be
broken and not replaced. Since hydrogen bonds are favourable interactions, there will be an
energy cost to putting non-polar molecules into water. Water therefore forces these molecules
out of solution to minimise the surface of contact and thus the number of hydrogen bonds which
are broken. Such forces known as hydrophobic forces are among the most important in driving
proteins to fold into compact structures (globular) in water. Also, these forces are responsible to
make different proteins assemble together to form structures found in muscles, membranes and
UNIT
33
other organs. In proteins therefore, hydrophobic regions are preferentially located away from
the surface of the molecule and form the interior core of the protein.
34
UNIT
Fig. 5. Charge relay transfer in chymotrypsin. R2 = aromatic amino acid; R1 = any other amino
acid.
Certain organophosphate compounds can selectively react with an acidic serine residue thereby
knocking off enzyme activity. Nerve gas which was unfortunately used in the first world war had
volatile serine alkylating compounds which inactivates the brain enzyme acetyl choline esterase
UNIT
35
36
1.
2.
The Hb and scHb are digested with the proteolytic enzyme trypsin which cleaves the
protein after basic amino acid residues Arg and Lys.
3.
Two separate strips of Whatman filter paper are spotted with Hb and scHb tryptic
peptides and the peptides allowed to separate using the technique of paper
electrophoresis at pH 2.0. Highly charged peptides will migrate more towards the
anode/cathode.
4.
The paper strips are dried, attached to larger squares of Whatman paper and
chromatographed at right angles to the electrophoretic direction using a solvent
system Butanol: Water:Acetic acid. In such a system peptides will separate based on
their partition coefficient between the solvent and paper which is dependant on the
relative hydrophobicity of the peptides. More hydrophobic peptides will move with
the solvent to longer distances.
UNIT
5.
The chromatograms are dried and stained with a suitable visualisation reagent like
Ninhydrin wherein peptide containing regions appear as orange yellow spots.
6.
The peptide map for Hb and scHb are compared and it was found that one peptide was
differently placed in the scHb map.
7.
On examining this peptide and determining its amino acid sequence, Ingram found
that it had a valine substitution for glutamic acid in the peptide.
The single substitution of valine for glutamic acid (val/glu are at the 6th position of
the haemoglobin beta chain) dramatically changes the structure of scHb making it
form fibres within the RBC resulting in the deformation of the cell (sickling). Since the
disease was due to a molecular alteration the term molecular disease was applied.
Peptide mapping became a useful technique to compare similar proteins from different sources.
Slowly the information became too vast and computers were used to store this data into
databases so that homology searches could be made. The protein fingerprinting data has been
further augmented with new databases containing 2-D electrophoresis patterns of entire proteins
from a given cell type, a technique developed by O'Farrel.
Purify Haemoglobin
Normal RBC
Trypsin treatment
Hemoglobin
scHemoglobin
Paper Electrophoresis
Paper chromatography
Result : All peptides were similar from both
samples except one (marked blue).
Peptide sequencing
UNIT
37
(Fig. 7).
In simple electrophoresis, the mobility of proteins is due to their charge, which is pH dependant.
At its isoelectric pH (pI), a protein does not possess any charge and thus will not move in an
applied electric field. This feature is exploited in the technique of IEF, which separates proteins
on the basis of their different pI values. Usually IEF is performed in thin tube gels. A pH gradient is
set up within the IEF gel by the inclusion of polymeric buffers known as ampholytes. These, like
proteins have many positive and negative charges and hence varying pIs. Because of their smaller
sizes they move rapidly in an electrophoretic run setting up pH gradients when they come to rest
at specific distances from the anode/cathode when they have no net charge. A protein sample
from a cell or any other source is then electrophoresed within these tubes wherein the different
proteins separate and migrate to their pI zones. The tubes containing the separated proteins is
then laid on a SDS-PAGE slab gel and electrophoresis continued at right angles to the IEF direction.
In SDS-PAGE proteins separate on the basis of their size and hence at the end of this
electrophoretic run proteins are separated into 2-D patterns with high resolution as two
properties of the proteins have been exploited in their separation- charge and size. Proteins in
the gels are stained with silver stains or other highly sensitive dyes and can be scanned, and
pictures stored into computer databases for analysis.
38
UNIT
Isolation of a protein from a microbial culture, plant and animal sources involves various
separation techniques (refer Textbook of Biotechnology for Class XI). These steps are collectively
known as downstream processing. In spite of a large biodiversity of microbes we are restricted to
certain bacteria/organisms which can be used as a source of protein as well as for introducing
genes. These microorganisms are designated as "generally regarded as safe" (GRAS). GRAS listed
organisms are non-pathogenic, non-toxic and generally should not produce any antibiotics.
Similarily, plant tissue derived enzymes which have application in the food industry must be
obtained from only non-toxic, edible plant species. One of the best known industrially useful
enzymes is papain obtained from the latex of the green fruit and leaves of the Papaya tree. This
enzyme finds application in meat tenderisation, clarification of beverages, digestive aids and
wound cleaning solutions.
The existence of slaughter house facilities in which large number of animals are regularly
processed to provide meat has also facilitated the collection of significant quantities of a
particular tissue required as a protein source. Insulin is a classic example of a peptide hormone
obtained from the pancreas of cows and pigs till the 1980's. Classical biotechnology required the
slaughtering of over 100 pigs or 15 cows to meet the insulin requirements for one diabetic person
for about one year. A rough estimate puts the number of diabetics in India by the year 2020 as 20
million! Obviously the requirement of insulin cannot be met by slaughtering pigs and cows.
Fortunately, the advent of genetic engineering has ensured the availability of recombinant
human insulin expressed in bacteria. Attempts are on to create transgenic animals by direct
micro-injection of DNA into ova or stem cells and produce insulin and other proteins in milk on a
commercial scale. This technology is called Molecular Pharming (Producing pharmaceuticals
using genetically modified plants or animals). Advantages of producing recombinant proteins in
milk are:
1.
2.
3.
4.
Some medically useful peptides such as oxytocin have also been produced by direct chemical
synthesis. In spite of different sources of proteins, the general principles of purification are
similar. The overall approach and techniques are outlined in Unit-II, Chapter-III of the class XI,
Textbook of Biotechnology. The exact details of the purification scheme for any given protein will
depend upon a number of factors such as:
1.
UNIT
Exact source material chosen and location of the target protein (intracellular or
extracellular).
39
2.
3.
18
15
=1.57 x 10-18 m3
2.007 x1015 cells (see previous answer) would have a volume of 2.007 x 1015 x 1.57 x 10-18 m3
= 3.15 x 10-3 m3 = 3.15 L Answer (a)
(1L = 10-3 m3)
Answer (b) 100% concentration
= 3.15 L
Therefore 5% concentration
= 100/5 x 3.15 = 63 L
Volume of the fermentor required would be more than 63 L (30% extra space) about 82 L.
The source material chosen will dictate the range and type of contaminants present in the
starting material. If the protein is extracellular, then one needs to separate the cellular
components and process the medium to isolate the protein of interest. However if the protein is
intracellular then the choice of method of cell disruption will depend on the cell type. Plant and
fungal cells require harsher breakage methods; animal cells are easier to break because of no cell
wall. Bacterial cells being very small require high pressure techniques. Once the proteins are
40
UNIT
released into suitable buffered solutions a variety of physico-chemical techniques are applied to
selectively purify the protein of interest from the others.
Genetically engineered proteins are often tagged with certain molecules in order to confer some
very pronounced physico-chemical characteristics on the protein of interest. This renders its
separation from contaminants more straightforward. The ability to detect and quantify the total
protein levels is an essential pre-requisite to the purification and characterisation of any protein.
A typical purification scheme can be analysed as follows (Table 1).
Table 1. Typical purification table
Procedure
Crude extract
20,000
40,00,000
200
Precipitation (salt)
5,000
30,00,000
600
Precipitation (pH)
1,000
10,00,000
1000
Ion-exchange chromatography
200
8,00,000
4000
Affinity chromatography
50
7,50,000
15,000
6,75,000
15,000
Note that the last column is a good indication of whether a purification step is useful or not. This is
because as a protein is purified its specific activity increases because the denominator should
ideally decrease as irrelevant proteins are removed and only the specific protein/enzyme of
interest is concentrated. Hence from the given table it is apparent that the step following affinity
chromatography, size exclusion chromatography, is redundant as the specific activity does not
change. For activity measurements it is also important to choose a proper assay method
reflecting the sensitivity required and further the method should be specific. In the case of
proteins absorbance measurements at 280 nm is easy, fast and non-destructive procedure for
monitoring the concentration.
Bioassays can sometimes be more sensitive than chemical assays. However one needs suitable
standards of known bioactivity values to arrive at a correct activity of the unknown sample.
Where a protein of biological interest is concerned, example insulin, bioassays are mandatory. If
the sample particularily has to be injected other safety tests such as toxicity have to be
performed.
Downstream Processing
After cells (bacterial, animal or plant) have grown to their requisite capacity in a fermentor it
becomes necessary to harvest the cells or medium depending in which component the
UNIT
41
recombinant protein is expressed and then purify the protein from other substances. These
processes are part of downstream processing and because of the large amounts of source (a
fermentor can be more than 1000 L capacity) bulk separation methods are used which are
different from laboratory scale purification although principles involved are similar.
In the case of intracellular microbial proteins, cell harvesting is done by filtration or
centrifugation from the fermentation medium, followed by re-suspension of cells in buffer or
water with subsequent cell disruption. Most proteins obtained from plant and animal tissues are
intracellular in nature. The initial step involves collection of the appropriate tissue, for example
collection of blood to obtain proteins, collection of pituitary glands to obtain pituitary hormones
etc.
42
UNIT
All efforts should be made to maximise protein stability during various steps. Some of the general
conditions which may be followed are:
1.
2.
3.
4.
5.
Bulk purchase of chemicals and other raw materials would bring down costs.
2.
Most large scale process equipment such as holding vessels and transfer pumps are constructed
from stainless steel or plastics, such as polypropylene. Glass vessels, so commonly used in
laboratory scale culturing techniques are seldom used for large scale preparatory work. Materials
used for large scale culturing must be inert and resistant to the corrosive action of any chemical
used during the process. They should not allow any leaching of potentially toxic metals or
chemicals into the product stream. It is useful to remember that any commercial plant has to
have good GMP (good manufacturing practice) . Any downstream processing requires the approval
of a regulatory authority in the form of a license to produce and market proteins designed for use
in the food or health care industry. This ensures that the processing procedures are based upon
established, validated methodologies. Generally 80% of the overall production cost are due to
steps in downstream processing and quality assurance.
A generalised downstream processing scheme used in the production of bulk protein/enzyme
from microbial sources is given in Fig. 9. Similar steps can be applied for animal and plant
sources.
UNIT
43
Fermentation (submerged/
semisolid culture system)
Microbial source
Intracellular protein
Recovery of cells
(centrifugation/filtration)
(centrifugation/filtration)
Removal of cells
Disruption of cells
(homogenization)
(centrifugation/filtration)
(centrifugation/filtration)
Purification of the protein
(precipitation, ion exchange, gel permeation
chromatography, affinity chromatography
Concentration of purified extract
(precipitation/precipitation)
Incorporation of stabilizes
Preservatives etc. Adjustment
To require biopotency
2.
Fingerprinting.
3.
4.
Protein sequencing.
5.
Mass spectrometry.
44
UNIT
Mass spectrometry
Mass spectrometry (MS) has emerged as an important tool in biotechnology. It is extremely useful
in obtaining protein structural information such as peptide mass or amino acid sequences. It is
also useful in identifying the type and location of amino acid modification within proteins. One of
-12
the major attractions of mass spectrometry is that as little as picomoles (10 ) of a protein sample
can be analysed. A mass spectrometer is an analytical device that determines the molecular
weight of chemical compounds by separating molecular ions according to their mass/charge ratio
(m/z) ratios. The molecular ions are generated either by a loss or gain of a charge (e.g. electron
ejection, protonation or deprotonation). After the ions are formed they can be separated
according to their m/z ratio and finally detected. The process of ionisation, ion separation and
detection in a mass spectrum can provide molecular weight or even structural information. A
sample M with a molecular weight greater than 1200 D can give rise to multiple charged ions such
as (M+nH)n+. Proteins/peptides have many suitable sites for protonation as all the backbone
amide nitrogen atoms could be protonated theoretically as well as certain amino acid side chains
such as lysine and arginine which contain primary amine functional groups.
A schematic diagram of the various parts of a mass spectrometer is indicated in Fig.10. Basically
a vapourised sample of a protein or peptide is introduced into the instrument wherein it
undergoes ionisation. The charged molecules are then electrostatically propelled into a mass
analyser (filter) which separates the ions according to their m/z ratio. The signal received upon
detection of the ions at the detector is transferred to a computer which stores and processes the
information.
ACCELERATION
IONISATION
electromagnet
to vacuum
pump
vaporised
sample
DEFLECTON
DEFLECTON
amplifier
chart
recorder
UNIT
45
The goal of mass spectrometric analysis of biomolecules like peptides and proteins is to create
gas phase ions from polar charged molecules which are generally non-volatile. A popular method
called Matrix Assisted Laser Desorption Ionisation (MALDI) is used to volatalise and protonate
peptides and proteins. In this procedure, the sample is transferred from a condensed phase to a
gas phase with the help of a solid matrix. Ion formation in MALDI is achieved by directing a pulsed
laser beam onto a sample suspended or dissolved in a matrix. The matrix plays a key role in this
technique by absorbing the laser light energy and causing the matrix material to vaporise. In the
gas phase, the matrix plays a role in sample ionisation. The charged molecules are directed by
electrostatic lenses from the ionisation source to the mass analyzer.
Multiply charged ions on analysis show patterns as indicated in Fig. from which molecular mass
can be deduced as also indicated in the legend below Fig. 11.
relative
intensity
(%)
5+
4001
0
5000
2+
10001
3+
6668
4+
5001
10000
[M+5H]5+
[M+4H]4+
m/z = 20005/5
m/z = 20004/4
[M+3H]3+
15000
[M+2H]2+
1+
20001
20000
[M+1H]1+
m/z = 20001/1
Fig.11. The same protein with a molecular weight of 10,000 contains 5, 4, 3, 2 and 1
charges. The mass spectrometer detects the protein ions at m/z = 2001, 2501, 3334, 5001
and 10,001 respectively.
Typically for protein identification, a crude extract is separated on a 2D gel. After visualising the
proteins on the gel, protein spots are excised and used for mass analysis by the MS technique
described above. For doing so the protein in the 2D gel after extraction from gel is either used
intact or it is cleaved into small peptides with a protease like trypsin which makes the mass
analysis easier. These peptides are separated on an on-line liquid chromatography system before
introduction into the mass spectrometer. Liquid chromatography techniques like ion exchange,
affinity or reverse phase column chromatography can be used to separate the peptides. These
peptides are either sequenced directly or the mass of peptides is analysed using database
searches (see Bioinformatics unit). With the human genome sequencing project, it is now possible
to identify new proteins by combining the mass spectrometric information with the genomic
46
UNIT
2.
3.
4.
Regulatory factors.
5.
Analytical application.
6.
Industrial enzymes.
7.
8.
Nutraceutical proteins.
UNIT
47
facilitated the development of modified forms which are faster acting like humulin. Humulin acts
in 15 min unlike pig insulin which takes 3 hours. Another growth factor- platelet derived growth
factor has been approved for diabetics who develop skin ulcers. Several other growth factors are
under various stages of clinical trials.
Regulatory factors
Several new regulatory factors were discovered that did not fit the classical definition of a
hormone. Initially they were known as cytokines. These include interferons, interleukins, tumor
necrosis factor and colony stimulating factors. The interferon family of INF alpha, beta and
gamma have found widespread therapeutic application; interferon alpha is used for treatment of
Hepatitis C, beta for Multiple Sclerosis and gamma for Chronic Granulomatous disease.
Analytical applications
Enzymes and antibodies have found a range of analytical applications in the diagnosis of diseases;
hexokinase for quantitative estimation of glucose in serum, uricase for uric acid levels in serum,
horse radish peroxidise and alkaline phosphates in ELISA etc.
Industrial enzymes
Proteolytic enzymes constitute an 8000 crore annual market for industrial enzymes. They find
application in the beverage industry, detergent industry, bread and confectionary industry,
cheese production, leather processing and meat industry. Alcalase is an enzyme used in the soap
industry, papain is used in the beverage industry, glucose isomerise in the confectionary industry
and chymosin is used in the cheese industry.
48
UNIT
TABLE : 2
Functional Property
Mode of action
Food System
Whipping/Foaming
Emulsification
Gelation
Viscosity
Water binding
Solubility
Protein solvation
Beverages
Browning
Flavour/Aroma
Nutraceutical Proteins
Nutraceutical is a word coined from combination of nutrition and pharmaceuticals. It has been
observed that several nutritional proteins also have therapeutic functions. For example whey
protein concentrates, lactose free milk (for lactose intolerant babies) and infant food
formulations.
Where does one get the raw building materials such as amino acids needed to make all body
proteins? During infancy we depend on milk. Baby milk formulations are also there (Amul,
Lactogen etc.) which have been formulated to have similar composition as mother's milk. All
these food materials provide the essential components nutritionally for growth and development
during the first few months of our existence. A typical composition of milk from buffalo, human
and cow sources is given in the Table 3 from which baby milk formulations can be made to suit an
infant.
UNIT
49
buffalo
human
cow
Protein (g)
3.8
1.2
3.3
2.
Casein (g)
3.0
1.4
2.8
3.
Lactalbumin (g)
0.4
0.3
0.4
4.
Lactoglobulin (g)
0.2
0.2
0.2
5.
Fat (g)
7.5
3.8
3.7
6.
Lactose (g)
4.4
7.0
4.8
7.
100.0
71.0
69.0
8.
Calcium (mg)
203.0
33.0
125.0
9.
Phosphorous (mg)
130.0
15.0
96.0
112.0
43.0
103.0
From the Table 3 it can be observed that milk contains several proteins, carbohydrates, lipids,
vitamins, antibodies, minerals etc. It is interesting to note that human milk has nearly half the
amount of casein as compared to cow and buffalo. Besides use of milk as a nutritional source,
claims have been made to the effect that curd is beneficial in the management of some types of
intestinal infections according to our ancient Sanskrit scriptures dating back to 6000 BC. Since
time immemorial whey (liquid part of curds) has been administered to the sick for the treatment
of numerous ailments. In 1603, Baricelli reported on the therapeutic use of cow's or goats whey,
sometimes mixed with honey and herbs. The spectrum of illnesses treated with whey include
jaundice, infected skin lesions, genitor-urinary tract infections. Gallen and Hippocrates insisted
on a minimum daily drinking of one litre of whey. Using modern scientific research it has been
possible to explain these observations. Whey proteins result in the elevation of a tripeptide
glutathione(gamma-glutamyl cysteinyl glycine) in cells. This peptide is a reducing compound and
has a broad range of functions including detoxification of xenobiotics and protection of cellular
components from the effect of oxygen intermediates and free radicals. More recently curd has
also been used as a pro-biotic (administered with antibiotics) because it is a good source of
beneficial bacteria which can colonise the intestinal tract. Table 4 gives the useful components
of whey.
50
UNIT
- Lactoglobulin
Bovine Serum Albumin
Immunoglobulins
Lactoferrin
Lactoperoxidase
Protease peptones/polypeptides
Free amino acids
Urea
Glycomacropeptides
Growth Factors
Lactose
Sialic Acid
Lactic Acid
Sodium
Potassium
Calcium
Magnesium
Chloride
Phospphate
Sulfate
Citrate
Heavy Metals
Milk Fat
Globule
Free Fat
Lipoproteins
UNIT
51
proteins and enzymes in order to improve their properties for thermal and pH stability, solvent
tolerance and solubility, catalytic potency etc. Given below is an example which has been
successfully used in the detergent industry.
52
Codon222
% activity w.r.t
wild type
Codon222
% activity w.r.t.
wild type
Cys
138.0
Gln
7.2
Met
100.0
Phe
4.9
Ala
53.0
Trp
4.8
Ser
35.0
Asp
4.1
Gly
30.0
Tyr
4.0
Thr
28.0
His
4.0
Asn
15.0
Glu
3.6
Pro
13.0
Lie
2.2
Leu
12.0
Arg
0.5
Val
9.3
Lys
0.3
UNIT
UNIT
53
Biological value (BV) measures the amount of protein nitrogen that is retained by the body from a
given amount of protein nitrogen that has been consumed. It has been observed that the BV of
whey proteins is the highest compared to rice, wheat, soya and egg proteins. Another index of
protein value is the protein efficiency ratio (PER). PER is used as a measure of growth expressed
in terms of weight gain of an adult by consuming 1g of food protein. The PER value of the following
proteins are arranged in decreasing order- whey, milk, casein, soya, rice, wheat. The modern day
approach for overcoming the nutritional deficiencies of seeds would be to engineer genes that
would encode storage proteins with more of the nutritionally desirable amino acids either by
inserting additional amino acids or substituting existing amino acids with new ones. Attempts are
already being made on zein storage protein genes of maize to enhance its nutritional value.
Introduction of entirely novel proteins that are highly enriched in specific amino acids is also
being considered.
54
UNIT
Review Questions
1.
2.
3.
4.
Briefly explain how the serine residue in some enzymes can become acidic (reactive).
Also suggest how you can confirm that a serine residue is involved in the catalysis.
5.
Why is sickle cell anaemia called "Molecular disease"? How can sickle cell haemoglobin be
identified?
6.
What are the principles behind Isoelectric Focusing and SDS/PAGE techniques? Why is 2-D
electrophoresis better than single dimension electrophoresis?
7.
8.
9.
10.
11.
What is the principle of MALDI-TOF? What is its main use in protein studies?
12.
E. coli is a rod shaped bacteria about 2 m long and 1 m in diameter. The average density
of a cell is 1 .28 g/ ml. Approximately 13.5% of the wet weight of E. coli is soluble protein.
Estimate the number of molecules of a particular enzyme per cell if the enzyme has a
molecular weight of 100,000 and represents 0.1 % of the total soluble protein. (Answer:
1626 molecules per cell).
UNIT
55
References
1.
Protein: Biochemistry and Biotechnology by Gary Walsh (2002), John Wiley & Sons, Ltd.
2.
Practical Biochemistry: Principles and Techniques Edited by Keith Wilson and John Walker
(2000), Cambridge University Press.
3.
Biochemistry by Lubert Stryer, J.M, Berg and J.L. Tymoczko (2002). W.H. Freeman.
4.
Principles of Biochemistry by Albert L. Lehninger, D.L. Nelson and M.M. Cox (2000), W.H.
Freeman.
5.
Biochemical calculations by Irwin H. Segel (1985), John Wiley & Sons, Ltd.
6.
56
UNIT
CHAPTER
5.3.1. Introduction
The term "GENOMICS" was coined in 1986 by Thomas Roder, to describe the scientific discipline of
mapping, sequencing and analyzing genomes. H. Winkler in 1920 had coined the term genome to
implicate the complete set of chromosomal and extra chromosomal genes of an organism, a cell,
an organelle or a virus.
The field of genomics relies upon bioinformatics, which is the management and analysis of
biological information stored in databases. During the mid-1980s to late 1980s, researchers
started to use computers as central sequence repository, from where the data could be accessed
remotely. Later in the early 1990s, genomics was transformed from an academic undertaking to a
significant commercial endeavor, a course followed by bioinformatics a few years later.
In retrospect, the genomics really began with the conception of the Human Genome (HGP) in the
mid-1980s. In the United States, the Human Genome Project officially started on October 1,
1990, as a 15-year program to map and sequence the complete set of human chromosomes, as
well as those of several model organisms. The goal of sequencing an estimated three billion base
pairs of the human genome was ambitious, considering that few laboratories in 1990 had
sequenced just 100, 000 nucleotides. By 1993, the Human Genome Project had become an
established international effort. The strategy of this international project was to make a series of
maps of each human chromosome at increasingly finer resolutions. In this approach,
chromosomes were divided into smaller fragments that could be cloned and then the fragments
were arranged to correspond to their locations on a chromosome. After mapping, each of these
ordered fragments would be sequenced.
Progress in stages
J. Craig Venter, a researcher at the National Institutes of Health, and his colleagues~ in early
1990s devised a new way to find genes. Rather than taking the Human Genome Project strategy of
sequencing chromosomal DNA-one base at a time, his group isolated messenger RNA molecules,
copied these RNA molecules into DNA, and then sequenced a part of these DNA molecules to
create expressed sequence tags, or "ESTs." These ESTs could be used as handles to isolate the
entire gene. Venter's method, therefore, focused on the "active" portion of the genome, which
was producing messenger RNA for protein synthesis. The EST approach has generated enormous
sized databases of nucleotide sequences, and facilitated the construction of a preliminary
transcript map of the human genome. The development of the EST technique is considered to
UNIT
57
have demonstrated the feasibility of high-throughput gene discovery (screening of all possible
gene candidates from the EST library), as well as provided a key impetus for the growth of the
genomics industry. After the success of these projects, Craig Venter moved again to sequence
entire genomes.
Evolving approaches
He devised the "whole-genome shotgun strategy," which involves randomly breaking DNA into
segments of various sizes and cloning the fragments into vectors. Since the fragments are
randomly cleaved from the genome, they tend to overlap, and a genome assembly program is
used to fit contiguous pieces by matching overlapping ends. This method was validated by
sequencing the entire genomes of a few selected microorganisms.
This is how, the first set of whole genome sequences of the smallest genome Mycoplasma
genitalium and Haemophilus influenzae Rd were released. To analyze the data, several computer
programs had to be adapted to fast computers, Later several new programs were also written to
accomplish the task of sequence assembly. Craig Venter established an organization called "The
Institute of Genomics Research (TIGR)" located in Maryland U.S.A., and soon whole genome
sequences were determined for many other bacteria including those that live in exotic
environments such as hot temperature or deep sea vents. Several bacteria of medical importance
were also sequenced. During this time, several groups from Europe also initiated whole genome
sequencing of bacteria such as Mycobacterium tuberculosis and Bacillus subtilis at Pasteur
Institute. Generally, in Europe large consortiums (group of organizations in various countries)
were formed to complement each other's strengths.
The exciting commercial era of genomics began with the establishment of Celera Genomics that
was dedicated to sequencing the human and the mouse genomes, compared to microbial
genomes, the human genome is large ~ 3 x 109 bps and also contains lots of repeated sequences.
These repeated sequences present difficulties in sequence assembly because doubts arise with
regard to their true order of arrangement in the genome. The parts containing the genes were
somewhat easier to assemble. The problem of sequence assembly of repeats and of unique
sequences by the computer is akin to this example. Suppose you were blindfolded and asked to
pick two balls of different size from a lot of mostly identical balls, you would make several
attempts but end up with failures most of the time. Further, you may not be able to distinguish
one ball from another. However, if you were given the same assignment of drawing two different
balls from a lot balls of all different sizes and shapes, then there is a good possibility of you
picking up two different balls at much fewer attempts, perhaps the very first attempt itself may
be enough.
But the unraveling of the human genome sequence gave us a surprise. Initial EST sequencing had
led to an estimate of over 100,000 genes being present in the human genome. The genome
sequence however, revealed that there are only about 30,000 genes. This number is only twice
58
UNIT
that of the fruitfly Drosophila melanogaster, a simple organism compared to the immensely
complex human being. Possessing only twice the number of genes of fruitfly challenges us to
search for other explanations that underlie the complexity of the humans. It turns out that
humans can achieve this through combinations of the genes. You can understand this by an
example.
Suppose, a mechanic has a set of 20 or 30 tools, each one dedicated to carry out a specific task.
Then the mechanic can accomplish 20 or 30 tasks. However, if the same set of tools had flexible
parts, then the same mechanic can generate several 'new combinations of these tools to carry
out hundreds of tasks. Naturally through combinations, this mechanic will have more business,
earn more money and will be most sought after compared to using the dedicated 'one job specific'
tools.
Below, the sections on genomics explain the different branches of this exciting new area. With
the development of automated sequencing, it has been possible to sequence genomes of many
organisms. According to the latest list displayed at the NCBI (National Centre for Biotechnology
Information) site, there are 1409 complete genome sequences of bacteria and archaea, 40
complete genome sequences of eukaryotes and 2537 complete genome sequences of viruses. The
sequencing projects have shown several interesting and unexpected findings. The term genomics
itself has undergone expansion in last few years and in the present context also includes genome
function. Genomics can be broadly divided into structural genomics and functional genomics.
Structural Genomics
Structural genomics primarily involves high-throughput DNA sequencing followed by assembly,
organization and management of DNA sequences. It represents an initial phase of genome
analysis, which involves the construction of high-resolution genetic, physical or transcript maps
of the organism. The ultimate physical map of an organism is its complete DNA sequence.
Although, in the last few years with the completion of several genome-sequencing projects, the
term structural genomics has also undergone transition. Several structural genomics initiatives
now encompass systematic and high-throughput determination of three-dimensional structures
of all proteins. The information and reagents provided by structural genomics are used to design
global (genome-wide) experiments to identify functions of proteins.
Functional genomics
Functional genomics represents a new phase of genome analysis and deals with the
reconstruction of the genome to determine the biological function of genes and the interactions
between genes. The fundamental strategy in a functional genomics approach is to expand the
scope of biological investigation from studying single genes or proteins to studying all genes or
proteins at once in a systematic manner. Functional genomics is therefore characterized by highthroughput or large scale experimental methodologies combined with statistical and
computational analysis of results.
UNIT
59
First it provides a means for the discovery of all the genes and thus provides an
inventory of genes.
Fourth, sequencing provides an index to draw and organize all genetic information
about the organism.
Fifth, and very important over time, is that the whole genome sequence is an archive
for the future containing all the genetic information required to make the organism.
There are several methods for small-scale sequencing, although most of these do not scale well to
sequence entire genomes. The two main methodologies used for genome sequencing are
discussed here. These have also been briefly discussed in the introduction.
60
UNIT
some are similar to each other in parts called overlapping parts, whereas, a few may be just
unique. After we feed all these data into a computer program, these sequences are joined by
finding overlapping parts. The result is, we get long pieces of DNA sequences. This process of
assembling continues until all overlapping parts are exhausted. Finally, we would get a large
portion of the genomic DNA sequence.
Even though in theory, the entire genomic DNA sequence can be obtained in this way, in practise,
this is not so. Some gaps in genomic DNA sequence do arise and these gaps need to be closed by
specific cloning of those regions and additional sequencing.
No. of
Genome size
chromo in base pairs
somes
The Number of
Predicted
genes
Part of the
genome
that
encodes for
protein
500,000
5000
90%
Yeast Saccharomyces
cerevisiae
16
12,068,000
6340
70%
100,000,000
19,000
27%
175,000,000 196,000,000
13,600
20%
157,000,000
25,498
20%
23
3,000,000,000
One of the surprises is the relatively small number of genes in a human genome ( 20,000 - 25,000
genes) in comparison to worm (19,000 genes). In fact some experts still think that there must be
at least 40,000 - 50,000 genes in the human genome, and that 30,000 just reflects the
UNIT
61
unreliability of in silico (i.e., computational) gene prediction. Still, it seems that there is no
simple correlation between the intuitive complexity of an organism and the number of genes in its
genome.
How to predict
response to drugs using
a SNP map
Fig.1. Definition of SNP, and an illustration to show how physicians can use SNP map to
determine how patients are likely to respond to a particular drug.
The vertical bars indicate various SNPs on the human DNA.
However, the other way to look at it is that the 0.2% difference in DNA sequence is enough to make
each individual unique. It is understood that on an average one in a thousand nucleotides are
different in genomes of two different individuals. Particularly, important variations in individual
genomes are the single nucleotide polymorphisms or SNPs, which can occur both in coding and
non-coding regions of the genome. It is believed that SNPs occur at 1.6 million to 3.2 million sites
in the human genome, and may affect gene function, depending upon exact base change and
where it occurs. It would be interesting to note the following research based observation.
62
UNIT
1.
2.
However, not all genetic variations are beneficial (see Table 2). Genomic variations
underlie differences in our susceptibility to, or protection from all kinds of diseases.
The severity of illness and the way our bodies respond to treatments are also
manifestations of genetic variations. For example, a single base difference in the
ApoE gene is associated with Alzheimer's disease, and that a simple deletion within
the chemokine-receptor gene CCR5 leads to resistance to HIV (Human
Immunodeficiency Virus) infections and the development of AIDS (Acquired
Immunodeficiency Syndrome). SNP analysis is therefore important for diagnostics and
a SNP database has been developed to aid these applications. An example of how a
physician can decide if a medicine prescribed will be effective to a patient is
illustrated in Fig. 1 (vertical bars denote various SNPs in patients' genomes).
Table 2.
1.
2.
3.
Migraine
1.
1.
2.
3.
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63
3.
Physicians can use patients DNA sample to determine the pattern of SNP genotype
profile and from that they can predict how patients are likely to respond to a
particular drug. SNP analysis can also be used in population genetics, as some SNPs
vary in different frequencies between populations.
4.
The genome sequencing projects have revealed that the genomes of organism
otherwise quite different in appearance are quite similar for example mouse and man,
are quite similar. Another example is that, among the conserved elements between
different species such as the worm and the yeast, substantial portion belongs to
genomic regions coding for proteins
5.
It is estimated that the difference between human and chimpanzee genomes is only 1
to 3%, while human and mouse share about 97.5% of their working DNA. These
similarities suggest that none of these genomes has changed much since we shared a
common ancestor 100 million years ago.
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UNIT
Unlabelled
DNA
5'
3'
3'
5'
Enzyme DNasel
DNA nicked
in random
locations
5'
3'
3'
5'
5'
3'
3'
5'
Newly
Synthesized DNA
Fig. 2. Nick Translation. Nicks (breaks) are created in the DNA using DNase I. Subsequently, DNA
Polymerase I synthesizes new DNA using the older one as template and incorporates the labelled
nucleotides in the process. Finally we get labelled DNA.
The DNA fragment size with fluorescence probe after nick translation depends upon the amount
of enzyme and the incubation time of reaction. The size range can be 300 to 3000 bp. The
application of FISH can be illustrated by taking an example of chronic mylogenous leukemia
(CML). It was observed from the karyotype analysis of the lymphocyte preparation made from
blood samples of CML patients that there was a 9-22 translocation in the chromosome (also called
'Philadelphia chromosome'). Although by counting the number of such cells it was possible to find
out the severity of the disease, it was not an easy procedure. The regions on the chromosomes
involved in translocation were identified on chromosomes 9 and 22. From the DNA library it was
possible to pick up clones carrying the particular genes involved in CML. Using nick translation it
was possible to flourescently label chromosome 9 region with red colour and chromosome 22
region with green colour and prepare the probe (Fig. 3).
UNIT
65
bcr
Ph1
abl
der9
It was observed that when CML lymphocytes smear cells were hybridized with the two probes
in situ and when observed under fluorescent microscope, the cells, which were affected,
appeared yellow (mixing of green and red colour produces yellow colour). The unaffected cells
appeared as red and green (Fig. 3). This technique known as Fluorescence in situ Hybridization
(FISH) allows knowing the status in the interphase unlike in karyotyping where you need a
metaphase chromosome. The status of the disease could easily be identified by counting the
number of cells, which appeared yellow. Further, it was possible to monitor the effect of
chemotherapy and drugs by taking out samples and counting the number of cells appearing
yellow.
66
UNIT
Microarray Technology
It is widely believed that thousands of genes and their products (i.e., RNA and proteins) in a given
organism function in a complicated and orchestrated way that creates the mystery of life.
However, traditional methods in molecular biology generally work on a "one gene - one
experiment" basis, which means that the throughput is very limited and the "whole picture" of
gene function is hard to obtain. In the recent years, a new technology, called DNA microarray, has
attracted tremendous interests among biologists. This technology promises to monitor the whole
genome on a single chip so that researchers can have a better picture of the interactions among
thousands of genes simultaneously.
Principle: Microarrays consist of large numbers of DNA molecules spotted in a systematic order on
a solid substrate, usually a slide (Fig. 4). The base pairing or hybridization is the underlying
principle of DNA microarray. Microarray exploit the preferential binding of complementary
single-stranded nucleic acids. A microarray is typically a glass (or some other material) slide, onto
which DNA molecules are attached at fixed locations (spots). The type of molecule placed on the
array units also varies according to circumstances. The most commonly used molecule is cDNA, or
complementary DNA, which is derived from messenger RNA. Since cDNA are derived from a
distinct messenger RNA, each feature represents an expressed gene. In order to detect cDNA
bound to the microarray, they must be labeled with a reporter molecule that identifies their
presence. This technique of introducing fluorescent dyes in DNA and its use in detection of target
molecule by hybridization has been previously applied in fluorescent in situ hybridization (FISH).
Procedure: Comparative hybridization experiments compare the amounts of many different
mRNA in two cell populations. If one wanted to compare a normal cell and a cancerous cell, the
following experiments are needed to be carried out. mRNA is first purified from total cellular
contents. mRNA accounts for only about 3% of all RNA in a cell so isolating it in sufficient quantity
for an experiment (1-2 micrograms) can be a challenge. Since free RNA is quickly degraded, to
prevent the experimental samples from being lost, they are reverse transcribed back into more
stable DNA form. The products of this reaction are called complementary (cDNA) because their
sequences are the complements of the original mRNA sequences. The reporters currently used in
comparative hybridization to microarrays are fluorescent dyes (fluors), represented by the red
and green circles attached to the cDNAs in Fig. 4. A differently-colored fluor is used for each
sample so that we can tell the two samples apart on the array.
The labelled cDNA samples are called probes because they are used to probe the collection of
spots on the array. The two cDNA probes are tested by hybridizing them to a DNA microarray. The
array holds hundreds or thousands of spots, each of which contains a different DNA sequence. If a
probe contains a cDNA whose sequence is complementary to the DNA on a given spot, that cDNA
will hybridize to the spot, where it will be detectable by its fluorescence. In this way, every spot
on an array is an independent assay for the presence of a different cDNA. Microarrays are made
UNIT
67
from a collection of purified DNA molecules typically using an arraying machine. The choice of
DNA to be used in the spots on a microarray determines which genes can be detected in a
comparative hybridization assay. The end product of a comparative hybridization experiment is a
scanned array image. A small piece of such an image is shown in Fig. 4.
Interpretation: The measured intensities from the two fluorescent reporters have been falsecoloured red and green and overlaid (in reality, you observe colours when the fluors are
stimulated by a laser). Spots, whose mRNA is present at a higher level in one or the other cell
population show up as predominantly red or green. Yellow spots have roughly equal amounts of
bound cDNA from each cell population. Therefore yellow spots correspond to genes expressed
approximately equally in both normal and cancerous cells whereas red spots correspond to genes
expressed in high amounts in normal cells. Similarly green spots correspond to genes expressed in
high amounts in cancerous cells. By drawing this distinction, we would be able to understand the
altered gene expression patterns in cancerous cells. This allows us to further understand the
mechanism and make attempts to develop cures.
Normal
Cells
Afected
Cells
mRNA extraction and
reverse transcription
Fluorescent labelling
of cDNA
Hybridization to a
microarray
Detection of Fluorescence
intensities through 2 color
channel laser
68
UNIT
This microarray technology promises to monitor the whole genome on a single chip so that
researchers can have a better picture of the interactions among thousands of genes
simultaneously. There are several names to this technology - DNA arrays, gene chips, biochips,
DNA chips and gene arrays. In the case of gene chips, the substrate for immobilization is a silicon
wafer and the probes are oligonucleotides spotted through photolithographic etching. In this
case of gene chip only 1 colour hybridization is performed per chip. Comparisons are done by
matching data from one chip to another through a special data normalization procedure. The
principle used in this technology is being extended to develop protein arrays also. This technique
has been used to study the following:
1.
2.
3.
4.
The strength of genomic studies lies in its global comparisons between biological systems.
Genomics studies provide initial guidelines to identify areas for deeper investigation and to see
how these results fit in the biological context.
5.3.6. Proteomics
The term Proteome refers to the complete protein set of a cell. Proteomics refers to the large
scale characterization of the entire protein complement of cells, tissues and even whole
organisms. Modern proteomic studies involve many different areas, which are illustrated in
Fig. 5. These include protein-protein interaction studies, protein function, and protein
localization. The growth of proteomics is a direct result of advances made in large-scale
nucleotide sequencing of various genomes. Without this development, protein identification
would have been difficult.
It is important to have information about the proteins simply because they are responsible for the
phenotype of the cells. It is impossible to understand mechanisms of disease, ageing etc. solely
by studying the genome. Only by understanding protein function and their modifications, drugtargets for various diseases can be identified. One of the major aims of proteomics is to create a
three dimensional map of a cell indicating the location of proteins.
The proteome of a given cell is dynamic. In response to internal and external cues biochemical
machinery of the cell could be modulated. This could lead to several changes in the proteins such
as post-translational modifications, changes in cellular localization, effect on their synthesis or
degradation. Thus examination of a proteome is like taking a snapshot of the protein environment
at a given time.
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69
It is speculated that no function can be assigned to about one-third of the gene sequences in
organisms for which the genome sequence is known. The complete identification of proteins in a
genome will help structural genomics projects. The aim of these projects is to obtain 3D structure
of all proteins in a genome. The structural analysis would be helpful in assigning function to many
of these proteins. In addition to identification of proteins, one of the major goals of proteomics is
to characterize post-translational modifications on proteins.
Signal transduction
Drug
targets
Proteome
mining
Disease mechanisms
Glycosylation
Expression Proteomics
Post-translational
modifications
Phosphorylation
Proteomics
Functional
Proteomics
Protein-protein
interactions
Structural Proteomics
Protein networks
Protein functions
Mouse knockouts
Organelle composition
Subproteome composition
Protein complexes
Fig. 5. Types of proteomics and the scientific knowledge that can be gained from them.
Types of Proteomics
Expression proteomics: The quantitative study of protein expression between samples that
differ by some variable is known as expression proteomics. Using this approach, protein
70
UNIT
expression of the entire proteome or of subproteomes between samples can be compared. This
could be useful in identification of disease specific proteins. For example: tumor samples from a
cancer patient and a similar tissue sample from a normal individual could be analyzed for
differential protein expression. Using two dimensional gel electrophoresis, followed by mass
spectrometry, proteins, which are over or under expressed in the cancer patient compared to the
normal individual can be identified. This could be compared with the microarray data (Fig. 4).
Identification of these could provide a lead in understanding the basis of tumor development.
Structural proteomics: Unlike comparing the same cell or tissue in normal and diseased state in
expression proteomics, structural proteomics are directed to map out the structure and nature of
protein complexes present specifically in a particular cellular organelle. The aim is to identify all
proteins present in a complex and to characterize all protein-protein interactions occurring
between these proteins. Isolation of specific sub cellular organelles or protein complexes by
purification can help assembling information about architecture of cells and explain how
expression of certain proteins gives a cell its unique characteristics.
Functional proteomics: Functional proteomics is a very broad term for many specific, directed
proteomics approaches. It can be defined as the use of proteomics methods to analyze the
properties of molecular networks involved in a living cell. One of the major objectives is to
identify molecules that participate in these networks. One of the successes of functional
proteomics is identification and analysis of protein networks involved in the nuclear pore
complex. This discovery has led to the identification of novel proteins which are important for
translocating important molecules from the cytoplasm of a cell to the nucleus and vice versa.
UNIT
71
Transcription
DNA
Translational
RNA
Translational
Regulation
Protein
Translational
Regulation
Proteolysis
Post-translational modifications
Localization
Alternative
spicing, mRNA
editing
Fig. 6. Processes through which genes can give rise to multiple protein products
with differing functions.
72
amino acids were not replaced at random but were altered with specific preferences.
For example, amino acids with similar physico-chemical characteristics were
preferred, one for another.
UNIT
some amino acids such as tryptophan, was generally not replaced by any other amino
acid.
This laid the first foundation for subsequent work on sequence comparisons using quantitative
approaches.
The National Biomedical Research Foundation (NBRF) compiled the first comprehensive
collection of macromolecular sequences in the "Atlas of Protein Sequence and Structure'
published from 1965-1978 under the editorship of Margaret O. Dayhoff. Dayhoff and her research
group pioneered the development of computer methods for the comparison of protein
sequences, for the detection of distantly related sequences and duplications within sequences,
and for the inference of evolutionary histories from alignments of protein sequences.
In 1980, the data library was established at the European Molecular Biology Laboratory (EMBL) to
collect, organize, and distribute nucleotide sequence, data and related information. Now its
successor is the European Bioinformatics Institute (EBI) located at Hinxton, U.K. The National
Centre for Biotechnology Information also started in USA as a primary information databank and
provider at about the same time. Later, the DNA Data Bank of Japan was initiated. The Protein
Information Resource (PIR) was established in 1984 by the National Biomedical Research
Foundation (NBRF) as a resource to assist researchers in the identification and interpretation of
protein sequence information. Today, all these databanks are in close collaboration with each
other and they exchange data on a regular basis.
As the sequence data began to accumulate rapidly, new powerful sequence analysis softwares
were needed. In parallel, firm mathematical basis was also required to develop algorithms.
Scientists from the field of mathematics, biology, and computer science entered the emerging
field of bioinformatics.
The databanks through their wide network of distribution of information are very important
sources for all researchers who take interest in asking fundamental questions in biology. Thus, a
major primary aim of bioinformatics is to spread scientifically investigated knowledge for the
benefit of the research community. Other aims include the development of softwares for data
analysis.
The word "bioinformatics" is a combination from biology and informatics. As it became clear that
biological polymers, such as nucleic acid molecules and proteins, can be transformed into
sequences of digital symbols informatics approaches can be used for analysis. Moreover, only
UNIT
73
limited set of letters is required to represent the nucleotide and amino acid monomers. It is the
digital nature of this data that differentiates genetic data from many other types of biological
data, and has allowed bioinformatics to flourish. Another key point is that the use of sequence
data relies upon an underlying reductionist approach: sequence implies structure which in turn
implies function. In the subsequent sections we will see the details of these activities.
Biological language
Letters
Nucleotide bases
Words
Sentence
Operons
Punctuation
Chapter
Chromosome
74
UNIT
Meaning
Base(s)
Guanine
Adenine
Thymine
Cytosine
G Or A
puRine
T Or C
pYrimidine
A Or C
Amino
G Or T
Keto
G Or C
A Or T
A Or C Or T
G Or T Or C
G Or C Or A
G Or A Or T
G Or A Or T Or C Any
The symbols, their meaning and the bases for the nucleic acid sequences are presented in
Table 3. The first 4 bases G,A,T,C, their symbols and the basis for nomenclature is clear. While
determining sequence data through experiments, sometimes, the sequence identity at a
particular position may not be clearly identifiable due to compression artifacts or other
secondary structure related problems. In most cases the problem can be solved by repeating the
experiment and also by sequencing the complementary strand. In a few cases, ambiguities may
persist. In such cases, the most probable results are inferred from the chromatograms.
For instance, at a position where the ambiguity is not resolvable between a 'G' or a 'C' but one can
be sure that there is no possibility of "A' or 'T' in the same position, then the symbol to be used is 'S'.
In most organisms, DNA is present as double stranded. The two strands are anti-parallel and
complementary to each other (following Watson-Crick base-pairing). However, the problem
UNIT
75
arises when we start encountering the symbols that mean more than one base at a given position.
Again, the IUPAC system comes to aid. The symbols to be used in the complementary strand
corresponding to the symbol at the same position in a given strand are specified in Table 4. In
certain cases, the complementary symbols are same as in the given strand because in both cases
they mean the same set of bases.
Table 4. Definition of complementary symbols.
Symbol
Complement T
The symbols and their meaning for the protein sequences are presented in Table 5. It is evident
that the number of symbols that mean more than one amino acid is very few.
Table 5. Symbol definitions for the amino acids.
76
Full name
Ala
Alanine
Arg
Arginine
Asn
Asparagine
Asp
Aspartic acid
Cys
Cysteine
Gln
Glutamine
Glu
Glutamic acid
Gly
Glycine
His
Histidine
Ile
Isoleucine
Leu
Leucine
Lys
Lysine
Met
Methionine
Phe
Phenylalanine
Pro
Proline
Ser
Serine
Thr
Threonine
Trp
Tryptophan
Tyr
Tyrosine
Val
Valine
Asx
Asx
Glx
Glx
Xaa
Xaa
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UNIT
77
to infer expression patterns by counting the number of ESTs corresponding to each gene divided
by the total number of ESTs.
GSTs : In Plasmodium falciparum the enzyme Mung Bean Nuclease (MNase) cleaves in between
the genes. A genomic DNA library generated by digestion with MNase was used for gene
identification in P. falciparum. The approach used was similar to ESTs. One read of sequence was
obtained from either ends. This data is referred to as genome sequence tags (GSTs). Usually,
genomic DNA sequence refers to the nuclear DNA.
Organelle DNA: Eukaryotic cells have organelles such as mitochondria and chloroplast. These
organelles have their own store house of information in the form of organelle DNA. Organelle DNA
codes for a few genes. The coding information for the rest of the genes reside in the nuclear DNA
of the same cell. If an organelle DNA has been sequenced the appropriate position in the
sequence submission form must be mentioned.
Other molecules: In addition to these molecules, the databases contain the sequences of other
molecules such as tRNA, and other small RNAs.
78
UNIT
(b)
Top scoring matches are ranked according to set criteria that serve to distinguish
between a similarity due to ancestral relationship or due to random chance. In most
analysis these criteria are not changed. However, if the user wishes, criteria can be
changed.
(c)
True matches are further examined thoroughly with other details accessible through
Entrez and other tools available at NCBI.
Note: Two sequences being similar does not mean that they are homologous. Homology is defined
as similarity due to common ancestry. Two sequences each from species A and species B are said to
be homologous if they have descended from a common ancestor to species A and species B.
Duplicated genes within a genome also may have similarities but these are referred to as
'paralogs'. Homologues will have the same function whereas paralogs may differ in functions.
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79
Homologene is a database of orthologs and homologs for several organisms like human, mouse,
rat, zebrafish and cow genes represented in UniGene and Locus Link. It is easy to infer
homologous relations using this database. RefSeq is a curated database of mRNAs and proteins of
organisms like human, mouse and rat. The data provided in RefSeq has been used in many cases
such as designing gene chips and describing the sequence features of the human genome.
There are many other resources provided by the NCBI. Discussing all of these is not possible within
the space limitations. Now, we mention a few other databases of importance to bioinformatics
work (see Table 6 below)
Table 6. Examples of other useful databases for Bioinformatics.
Database
Information available
PALI database
Curator: A curator is one who reviews and checks newly submitted data ensuring all mandatory
information has been provided, that biological features are adequately described and that the
conceptual translations of any coding regions obey known translation rules. This process is called
curation.
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UNIT
Regulatory sequences: Regulatory sequences can also be identified and analysed by using
bioinformatics tools.
Inferring phylogenetic relationships: Information regarding the relationships between
organisms can be obtained by aligning multiple sequences, calculating evolutionary distance and
constructing phylogenetic trees.
Making a Discovery: Using the bioinformatics tools and databases, the functions of unknown
genes can be predicted.
Review Questions
1.
2.
3.
What are the IUPAC codes for (i) 'G' or 'C', (ii) A or T, (iii) A or C, (iv) C or T, (v) A or G
4.
What are the conventions adopted by the Database personnel to store nucleic acid data and
protein sequence data with regard to the direction of the sequence? What is the basis of the
convention?
5.
What are the single letter IUPAC codes for alanine, glycine, tryptophan, tyrosine, serine,
methionine?
6.
What are the different types of molecules on which sequence data is obtained and
deposited in the database?
7.
8.
Suggest one possible way for going about analyzing a given sequence using bioinformatics.
9.
Using microarrays one can identify the genes expressed differently in normal vs cancer cell
types. Explain.
10.
What is random shotgun sequencing? What are the difficulties with assembling sequences
with repeats?
11.
What were the surprises revealed from genome sequencing? What underlies the accrual of
complexity in humans even though the number of genes are low?
12.
13.
14.
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81
References
1.
2.
Proteomics-from protein sequence to function (2002). S.R. Pennington and M.J. Dunn BIOS
Scientific Publishers Limited.
3.
Bioinformatics A practical Guide to the Analysis of Genes and Proteins (2002). A.D.
Baxeuarus and B.F Francis Ouellehe. A John Wiley & Sons, Inc.
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6
CELL CULTURE AND
UNIT
GENETIC MANIPULATION
CHAPTER
6.1.1. Introduction
In your previous class you have studied that microorganisms like bacteria and fungi are cultured
to obtain a number of products, which are beneficial for human beings. Some of the commonest
examples are antibiotics, ethanol and enzymes. With the advent of biotechnology and genetic
engineering techniques, microorganisms are also being used for the production of recombinant
molecules such as insulin, hepatitis B vaccine, growth hormones and interferons. Microorganisms
are also grown or cultured for production of biomass per se such as the single cell protein (SCP).
Besides these, scientists culture microbes in their laboratories for research proposes. It is
therefore very important to understand how microbes are cultured in the laboratory or in the
industry for production of various important molecules.
A microbial culture works as a factory in which the metabolism of a microorganism is exploited to
convert raw material into products. There is a maximal capacity for a single microbial cell to
convert raw material into products in a given period of time. Hence, to produce a given amount of
product within a given time, it is necessary to calculate the number of cells as well as doubling
time of the particular bacterium used in the process. Like any other chemical reaction, which
requires an appropriate temperature, pressure, pH and solvent, microbes also grow in an
appropriate environment of pH, temperature, nutrients (provided by the growth medium) and
the substrate (raw material), which is converted by the bacterium into the desired product.
This chapter deals with basic principles of culturing microbes in the laboratory, large-scale
industrial productions, and their applications. An important point that should be kept in mind is
that, the requirements for culturing microbes on a large or industrial scale are different from
culturing them in a laboratory.
UNIT
85
beef extract, yeast extract or casein digest. Such media are called semi-synthetic media. These
media are well suited for culturing most microbes in the laboratory. However, when one wants to
culture microbes on a large scale for production of useful metabolites, one uses sources of
nutrients, which are economical and available readily. In some cases, these nutrient sources or
raw materials may need a pretreatment before use. Other considerations while selecting the
growth medium are:
Should yield maximum product or biomass per gram of the substrate used.
Carbon sources: Cereal grains, starch, cane molasses, glucose, sucrose and lactose are
commonly used as carbon sources.
Nitrogen sources: Ammonium salts, urea, corn steep liquor or slaughterhouse waste are used as
nitrogen sources.
The carbon and nitrogen sources mentioned above are cheap and easily available.
Trace elements: Trace elements like Fe, Cu, Zn, Mn, Mo are also needed in small quantities.
Growth factors: Some microorganisms cannot synthesize specific nutrients like some amino acids
or some vitamins. Such growth factors are incorporated into the medium in adequate amounts as
a pure compound or as a component of complex mixture.
Antifoaming agents: In most microbiological processes, foaming is a problem. Foaming may be
due to the components of the culture medium or some molecules produced by the microbes. The
most common cause of foaming is the presence of proteins in the culture medium. Excess foaming
denatures proteins and provides hindrance to free diffusion of oxygen in the medium. Commonly
used antifoams are fatty acids, such as olive oil or sunflower oil. Silicones are also used as
antifoams.
Energy sources: The carbon sources such as carbohydrates, lipids and proteins in the culture
medium are the major sources, which provide energy for the growth of microbes.
Water: Water is the major component of the culture media. When required for culturing microbes
in the laboratory, single distilled or double distilled water would suffice. When assessing its
suitability for large-scale microbial culture as in industry, we should consider the pH and
dissolved salts. Water is also required for ancillary services such as heating, cooling and rinsing.
Clean water of consistent composition is therefore a prerequisite for large-scale cultivation of
microbes.
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UNIT
Culture Procedures
Sterilization procedures: When microbes are cultured in laboratory in 100 -1000 ml flasks,
sterilization of the nutrient medium can be easily done in an autoclave at a pressure of 15 pounds
psi (per square inch) for 15-20 minutes. However, when microbes are cultured on a large scale
such as for fermentation processes in the industry, we need to sterilize thousands of liters of
culture medium or the substrate. There is also the problem of sterilizing the huge culture vessel
i.e. the fermentor. Steam is used almost universally for sterilization of the fermentation media.
Medium may be sterilized in situ in the fermentor itself. But if the medium is sterilized in a
separate vessel, the fermentor must be sterilized before sterile medium is added to it. The
fermentor is sterilized by passing steam through its jacket or coils. The steam is also sparged into
the vessel through all entries allowing it to exit slowly from the air outlet. Steam pressure is held
at 15 lbs psi in the vessel for 20 minutes.
The air used in the fermentation process should also be sterilized. This is done by filter
sterilization.
Aeration and mixing: When microbes are cultivated in the laboratory, aeration and mixing can be
easily achieved by putting the flasks on shakers (shake culture). This may be further augmented
by the use of baffle flasks (Fig. 1). In large-scale bioreactors however, transfer of oxygen to
microorganisms is particularly difficult because the microorganisms must be well mixed and the
oxygen dispersed to achieve relatively uniform concentration. Many fermentor designs have
mechanical stirrers to mix the medium, baffles to increase turbulence, which ensure adequate
mixing. Forced aeration also provides mixing and the needed oxygen.
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87
Shakers: Continuous agitation of the culture medium also greatly improves the efficiency of the
oxygen transfer and this improves the growth of the microbes. In the laboratory, this is done by
the use of shakers (Fig. 2). Shakers may be end-to-end type or rotatory type. These may be
designed for use at the ambient temperature or in a controlled temperature environment
(incubator shaker).
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UNIT
pH controller
Steam
Acidibase reset
and pump
Exhaust
Impeller
Cooling
water
out
Cooling
jacket
Culture
broth
Cooling
water in
Sparger
(air bubbles)
Sterile air
Steam
Harvest
UNIT
89
90
UNIT
QS
QS
UNIT
91
equal volume of culture from culture vessel. That is, formation of new biomass by the culture is
balanced by the loss of culture from the vessel. In continuous culture, cells can be grown at a
particular growth rate for an extended period of time. Most of the time, the chemical
environment inside a continuous culture is constant. In a chemostat, constant chemical
environment is maintained whereas in a turbidostat constant cell concentration is maintained. If
medium is fed to such a culture at a suitable rate, a steady state is achieved eventually. At steady
state, the cell growth and substrate consumption takes place at a fixed rate (Fig. 7). Growth rate
of cells remains constant during steady state operation. This means that at steady state, the
concentration of cells, metabolites and other nutrients inside the reactor remain constant. That
is, formation of new biomass by the culture is balanced by the loss of the culture from the vessel.
Continuous culture is most suitable for production of biomass or metabolites. Continuous culture
is widely used for production of both microbial biomass as well as their metabolites.
QS
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UNIT
quantitative measurement of cell growth. The parameter that characterises microbial growth is
the doubling time. It is the time required for the cell mass or number to double its original value
during the balanced growth (i.e., log phase) of the organism.
Measurement of cell mass or number is one of the easiest ways to measure microbial growth.
(1)
It is carried out by measuring the dry weight of the cell material in a fixed volume of the
culture by removing the cells from the medium, and drying them till constant weight is
obtained. For E. coli, the bacterium that is extensively used in laboratories, the dry cell
weight of 1 billion cells is approximately 150 mg.
(2)
Apart from the methods described above, other methods are also used for measuring cell growth.
Some of these are: measurement of wet weight of cells, turbidity measurements, ATP
measurement, viable plate count (colony forming units or cfu) and use of Coulter counter.
Coulter counter is an electronic instrument and is used for direct counting of microbial cells in
suspension. In Coulter counter, the cells are made to pass in a single file through electrical field
and the electrical impedance thus generated is recorded.
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93
point exponential growth occurs and cell biomass or cell number increases at a constant rate.
Death
Stationary Phase Phase
Number of Microbes
Lag
Phase
Time
Fig. 8. A typical bacterial growth curve.
Mathematically, exponential growth can be described by two methods:
One related by biomass (X) and the other by cell numbers (N).
Let us first consider the exponential growth as related to biomass.
Balanced growth is like a chemical reaction where substrate is getting converted into product i.e.
cell biomass in this case. Thus, growth behaves like an auto-catalytic reaction and the rate of
growth will be proportional to the cell biomass present at that time. This can be described as
follows:
rate of change of biomass dX / dt = X
Eq.------- 1
where
94
: time (h)
-1
UNIT
The equation 1 can be rearranged to estimate the specific growth rate ( ) as:
The specific growth rate = 1 / X . dX / dt
Eq.------- 2
Specific growth rate is an index of rate of growth of the cells in particular environment. In other
words, specific growth rate is characteristic of the microorganism and is a function of the growth
environment including temperature, pH, medium composition and levels of dissolved oxygen.
During the period of true exponential growth, equation 1 can be integrated to provide the
following equation:
Xt = X0 e
Eq.
------- 3
where
Xt : biomass conc. after time t
X0 : biomass conc. at the start of the exponential growth
e : base of the natural logarithm
Taking natural logarithm:
ln Xt = ln X0 + t
Eq.
-------- 4
= (ln Xt ln X0) / t
Please note that if log10 values were plotted instead of natural log the slope would be equal to
/2.303.
= 2.303 (Log Xt - LogX0) / t
Eq.
-------- 5
If we consider a situation where at time zero, the cell biomass is X0, then after exponential growth
equivalent to one doubling time (td), the microbial biomass will become 2 X0.
i.e. Xt = 2X0
when t = td
UNIT
Eq.
----------- 6
95
td = ln 2
Thus,
td = 0.693 /
Eq.
---------- 7
No. of cells
Mathematically
No
No x 2
No x 2 x 2
No x 2 x 2 x 2
No 2
No 2
No 2
No 2
Thus, after a period of exponential growth time (t), the number of cells (Nt) will be given by:
Nt = N0 2 n
Eq.-------- 8
Where,
n = the number of the divisions
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UNIT
Eq.-------- --- 9
The mean generation time or doubling time (td) i.e. the time taken to undergo single generation
that doubles the population is:
td = Total growth time / number of divisions
= t/n
Eq.---------- 10
The specific growth rate and doubling times of the organism decide the medium requirements
and fermentation batch time for the production of biochemical molecules, and thus, are
important parameters for large-scale production process.
Example: Calculate the generation time (doubling time or td) of a bacterial population in which
the number of bacteria increases from 104/ml to 107/ml during four hours of exponential growth.
Answer: First calculate the number of divisions the population must have undergone to increase
from 104 to 107 in 4 hours.
Using the equation 9:
td = 240 minutes / 10
= 24 minutes
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97
the quantitative approach of producing just 1 liter of curd at home. Imagine the issues involved
when one has to produce thousands of liters of curd in dairy industry.
Direct production of microbes on a large or commercial scale has the risk of not only large
investments, but also producing products, which may not be of appropriate quality so that there
are problems in their commercialisation. To avoid these risks, the manufacturers try to validate
laboratory process on an intermediate scale before attempting commercial production. This step
is carried out in a pilot plant, which is a mini version of the commercial plant.
Before setting up a commercial scale plant the results, which are derived from pilot plant, are
theoretically extrapolated to the commercial scale in terms of equipment size (fermentor /
bioreactor), utilities like steam, water, electricity, fittings etc, labour (technical and nontechnical) and market surveys. All these data are compiled into a techno-economic report to
assess the feasibility of the project. Such a report is also important for seeking financial
assistance for the project from banks or other financial institutions.
The following example will give you an idea about the importance of scale-up calculations for the
bioprocess industry in terms of reactor size:
Recombinant insulin is produced at 100 mg/L by E. coli at a cell concentration of 1 g/L. Calculate
the volume of reactor (size of the fermentor) needed to produce 1 Kilogram of insulin in the
following conditions:
(a)
When the cell concentration is 1 g/L and insulin production is 100 mg /L.
(b)
When the cell concentration is 50 g/L and insulin production is 100 mg /L.
(c)
When the cell concentration is 50 g/L and insulin production is 500 mg /L.
Answer:
(a)
(b)
In this case the cell concentration is increased to 50 g/L; so insulin production per liter
will be 50 X 100 = 5000 mg = 5 g / L;
Thus, to produce 1 Kilogram of insulin we need 1 Kilogram / 5 g = 1000 g / 5g = 200 g.
So, if the cell concentration is increased 50 times, we need 200-litre reactor to
produce 1 Kilogram of insulin.
(c)
98
In this case cell concentration is high (50 g/L) and the insulin concentration is also
very high (500 mg/L). Thus insulin yield per liter of culture is 500 X 50 = 25, 000 mg / L
which is 25 gram/L. Thus, to produce 1 Kilogram of insulin we need 1 Kilogram / 25
UNIT
Most of the antibiotics are secreted into the medium, so their isolation mainly involves steps
UNIT
99
100
UNIT
depicted in Fig. 9.
Most of the recombinant proteins expressed in E. coli accumulate intracellularly as protein
aggregates. So their isolation and purification involve steps as described in Fig. 10. Once the pure
metabolite is obtained, a stabilized formulation is made using several ingredients known as
excipients.
Strain isolation:
The sample containing the microbes (e.g. soil) is put in a nutritive medium and allowed to grow in
shake cultures. The growth conditions (e.g. temperature) or nutrients in the medium are
provided such that these favour the growth of microbes of our interest. This is called enrichment
technique. The enriched culture can further be sub-cultured by taking a small inoculum and
putting it into fresh medium. In this way, the growth of the desired organisms improves
successively. Further screening is done using a method where the organism will show its desired
properties. For example, if we are looking for a microorganism, which produces an antibiotic, we
may detect it by growing the culture on an agar plate in the presence of that bacterium against
which antimicrobial activity is desired. Immunological methods are also available in which the
microbes producing products are detected using specific antibodies. Molecular biology has made
available a variety of probes, which enable the detection of organisms capable of producing
specific products. Recently some of these methods have been adapted to robotic automation
resulting in enormous throughput screening of microbes for newer / novel molecules.
Strain improvement:
Strain isolation procedure described above only identifies a strain, which has the capability or
potential to produce a desired molecule. It does not ensure that it produces molecule in sufficient
quantities to be economically viable. Techniques of classical genetics and genetic engineering are
used to improve the desirable characteristics of the strain.
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101
Mutation Selection: This is one of the oldest methods of strain improvement. The strain is
exposed to chemical (e.g. nitrosoguanidine or NTG) or physical (e.g. UV rays) mutagens and the
mutants having improved characteristics are selected. It is often necessary to carry out multiple
successive mutations before we get the desired results. One of the classical examples of strain
improvement using this methodology is the production of antibiotic penicillin. Several successive
mutations were necessary to develop a strain of Penicillium chrysogenum capable of producing
nearly 100 times the concentration of penicillin produced by the original strain (Penicillium
notatum), thus making production of penicillin commercially feasible.
Genetic Engineering Techniques: Until the recent breakthroughs in the techniques of genetic
engineering, a bacterium could produce only substances coded for in its genome. Genetic
engineering techniques about which you have learnt in the unit V (Chapter 1) allow totally new
properties or capabilities to be added to the microorganisms giving rise to recombinant strains.
Using these techniques, microorganisms may be manipulated to, synthesize or secrete enhanced
quantities of biomolecules, facilitate production of novel compounds or allow utilization of
cheaper substrates. Using these techniques, the microorganisms may also be utilized to produce
plant, animal or human proteins. Some of the valuable human proteins which are being produced
in microorganisms using this technology include recombinant human insulin (Humulin), hepatitis
B surface antigen, human growth hormone and interferons. These proteins can now be produced
in large quantities. Consequently the cost of the therapies which make use of these proteins viz.
insulin (diabetes), hepatitis B surface antigen (vaccination against hepatitis B virus), human
growth hormone (growth retardation) and interferons (immunotherapy) has been reduced
considerably.
The tools, which are used for genetic engineering viz. the restriction enzymes, cloning and
expression vectors and introduction of recombinant DNA into host cells, have been discussed
fairly in detail in the unit V (Chapter 1). However there are many practical problems, which must
be taken care of before a foreign (heterologous) gene may be expressed in a microorganism to
make it commercially viable. For example, when a foreign gene is introduced into a host
bacterium, it may not be expressed there. This problem is overcome by placing foreign gene
under regulatory controls recognized by the host microorganism. To maximize production of
foreign protein, the expression vector used is such that it replicates to high copy number and is
stable. The foreign gene should ideally be linked to a strong promoter that has high affinity for
RNA polymerase. The foreign gene may also be put under the control of a regulatory switch such
that production of recombinant protein does not occur until required.
When a eukaryotic gene (e.g., plant, animal, human) is expressed in prokaryotic (bacterial) host,
there are additional problems to be tackled. The non-coding region of eukaryotic gene must be
excised. This requires use of reverse transcription of mRNA into cDNA. Additionally, the
recombinant protein may not be secreted into the medium or its incorrect folding and
accumulation intracellularly may generate inclusion bodies. All these problems make
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UNIT
downstream processing difficult and costly. Thus, an alternative would be to use a eukaryotic
expression host. For this purpose, Saccharomyces cerevisiae has been quite popular because it is
safe and scientists have long experience of using this yeast in industrial fermentations. Detailed
information on biochemistry, physiology and genetics of this yeast is also known. Moreover, this
yeast can be manipulated genetically rather easily. However, product yields are relatively low at
1-5% of the total protein. Other yeasts like Pichia pastoris has a number of advantages: it has
strong inducible promoters; it is capable of making post-translational modifications similar to
those performed by human cells; downstream processing is simpler as Pichia does not secrete its
own proteins into the fermentation medium.
Metagenomics
In the last few years, another approach has been developed to identify and select microbial genes
synthesizing novel molecules. This approach directly utilizes the large number of microbial
genomes present in an environmental niche, for example in soil, in water such as ocean or in
human gut. These genomes are contributed by both the culturable and the non-culturable variety
of microbes and together constitute what has been termed as metagenome. The collective DNA is
extracted from a sample of soil, water or any other environmental niche. It is subjected to
restriction digestion using restriction endonucleses and the fragments are cloned as described in
unit V (Chapter 1) of this book. The clones are then screened for presence of a variety of
molecules. The clones expressing novel molecules or molecules with improved characteristics are
used for large-scale production by fermentation techniques described in this chapter.
The metagenomic approach not only give the scientists an opportunity to cast a wider net on
microbial resource present in the environment to fish out genes of their interest, it also gives
them the opportunity to analyze the genomes of the microbes without culturing these in the
laboratory. Thus, it is really a very useful approach to study those microbes, which are difficult to
culture in the laboratory or have never been cultured in the laboratory as yet, and analyze these
to see if they carry any genes, which may be exploited for human use. A typical procedure
depicting metagenomic approach is shown in Fig. 11.
UNIT
103
Strain preservation:
Once a strain producing a novel or desired product has been obtained, it must be appropriately
preserved for future use. If not done properly, the strain may be lost through loss of viability or
even show decline in the production of the product for which it was isolated.
o
Storage on agar: Cultures are grown on agar slants or stabs & stored at 5 to -20 C. These must be
sub-cultured at approximately 6-month interval. The time of sub-culture may be extended to 1
year if cultures are covered with sterile mineral oil.
Storage in liquid nitrogen: The culture is grown and a cryoprotective agent like glycerol (10-30%)
is added. These are dispensed in sealed ampoules & frozen in liquid nitrogen. (-176 to -196 oC).
Lyophilisation: Lyophilization or freeze-drying involves freezing of a culture followed by drying
under vacuum. This results in sublimation of cell water. Lyophilised culture may remain viable for
5-10 years or more.
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UNIT
Products
Saccharomyces cerevisiae
Ethanol
Aspergillus niger
Citric Acid
Penicillium chrysogenum
Penicillin
Streptomyces griseus
Streptomycin
Corynebacterium glutamicum
L-Lysine
Propionibacterium shermanii
Vitamin B12
Aspergillus oryzae
Amylases
Leuconostoc mesenteroides
Dextran
Escherichia coli
(via recombinant technology)
Saccharomyces cerevisiae
UNIT
Alcaligenes eutrophus
105
One of the most important uses of microorganisms in recent years has been as hosts for
production of recombinant proteins using genetic engineering techniques. Expression of human
insulin in Escherichia coli and hepatitis B surface antigen in Yeast for making hepatitis B vaccine
are the most notable examples of applications of microbes for human use. Some specific
examples of products derived from microbial cultures have been given in Table 1.
Toxicity and allergy associated with the use of microbially produced biomolecules
especially the recombinant molecules.
When the microbes are to be used in industrial processes, it is preferred to use only those
microorganisms, which are included in the 'GRAS' (generally regarded as safe) category. Also,
when microbes are to be used as hosts for production of recombinant molecules, this should be
carried out using GRAS organisms.
The main fear associated with the genetically engineered microorganisms (GMO) or recombinant
strains is that they could escape from the laboratory into the environment with unpredictable and
perhaps catastrophic consequences. It was believed that such released microorganisms could
upset the balance of nature or that foreign DNA in the new microorganism could alter the
metabolic activity of the host microbe in unpredictable and undesirable ways. Guidelines have
been established to ensure safe working practices and levels of containment based on potential
hazards. Many of the GRAS organisms found in nature have been genetically modified so that
these may be used as host for production of recombinant biomolecules. At present, all new
releases of genetically modified organisms are evaluated by expert committees on a case by case
basis for various safety aspects before approval for their use is granted.
Besides these, whenever we are required to culture microbes whether for research or for
industrial applications, we must follow good microbiological practices (GMP). These include:
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UNIT
i.
Persons must wash their hands with germicidal soap after handling viable
microorganisms.
i.
Eating, drinking, smoking etc. are not permitted in the working area.
ii.
Food is to be stored outside the work area in cabinets / refrigerators designated for
this purpose only.
iii.
iv.
All procedures are carried out in a way, so as to minimize splashes and generation of
aerosols.
v.
vi.
vii.
All cultures, stocks or other waste are decontaminated and autoclaved before
disposal.
Review Questions
1.
While culturing microbes in the laboratory in a flask, what measures do you suggest to
enhance their growth?
2.
Differentiate between:
a.
b.
3.
4.
Explain what is meant by steady state in relation to the growth of microbial cultures?
5.
6.
What problems must be tackled while expressing a eukaryotic gene in a prokaryotic host?
7.
How would you ensure that production of a recombinant molecule does not occur until
required?
8.
What problems make the downstream processing of recombinant proteins difficult and
costly?
9.
UNIT
107
10.
11.
12.
What is lyophilisation?
13.
14.
15.
Enlist five good laboratory practices, which need to be followed while working with
microbes.
16.
References
1.
Introduction to Biotechnology, By C.M. Brown, I. Campbell and EG. Priest, Panima Publishing
Corporation (2002).
2.
Industrial Microbiology, An introduction By M.W. Waites, N.L. Morgan, J.S. Rockey and G.
Higton, Blackwell Publishing (2007).
3.
Microbial Biotechnology, Principles and Applications By Lee Yuan Kun (Ed), World Scientific
(2004).
4.
5.
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UNIT
CHAPTER
6.2.1. Introduction
The plant cell culture is based on a unique property of the cell, i.e., totipotency which may be
defined as the ability of a plant cell to regenerate into whole plant on a defined artificial nutrient
medium under the suitable physical conditions in the laboratory. In fact, it was Schwann who first
drew attention to the fact that a single cell has the capacity to both grow and divide in a self
regulatory fashion and that a single cell is also totipotent. This was stated in the famous 'cell
theory' which was proposed in the beginning of 19th century by Schleiden and Schwann. Gottlieb
Haberlandt, who is regarded as 'Father of Plant Tissue Culture', first attempted in 1902 to
cultivate the mechanically isolated plant leaf cells on a simple nutrient medium. Though
unsuccessful in achieving the growth and differentiation of the cultured cells, he has made
several predictions in plant tissue culture like the concept of growth hormones, the use of embryo
sac fluids, the cultivation of artificial embryos from somatic cells, etc. and indeed all of his
predictions were found to be true as demonstrated by later researchers. In the first phase during
the period 1902 - 1930s, attempts were made by several scientists to culture the isolated plant
organs such as roots and shoot apices (organ culture). In the second phase during 1940s - 1970s,
the extensive studies were undertaken to develop the suitable nutrient media to culture plant
tissues, embryos, anthers, pollen, cells and protoplasts, and the regeneration of complete plants
(in vitro morphogenesis) from cultured tissues and cells. Since 1980s, a new era has started
involving the introduction of foreign genes into crop plants using cell and tissue culture systems to
develop genetically modified (GM) or transgenic crops with improved characteristics, which may
be responsible for the 'second green revolution.
Selection of suitable explants like shoot tip, leaf, cotyledon and hypocotyls.
2.
3.
Inoculation (transfer) of the explants onto the suitable nutrient medium (shoot
regeneration medium, which is sterilized by autoclaving or filter-sterilized to avoid
UNIT
109
Growing the cultures in the growth chamber or plant tissue culture room (Fig. 2A),
having the appropriate physical conditions [i.e., artificial light (16 h photoperiod),
temperature (~26oC) and relative humidity (50-60%)].
5.
6.
7.
Transfer of plants to the transgenic green-house (Fig. 2B) or field conditions following
the acclimatization (tissue hardening) of the regenerated plants.
Nutrient media
The in vitro culture of plant parts or cells require a variety of nutrients and suitable physical
conditions, unlike the intact plants which can synthesize their own food and many other essential
compounds needed for their growth and development using light, CO2, water and minerals. The
composition of plant tissue culture medium can vary, depending upon the type of plant tissues or
cells that are used for culture.
The typical plant tissue culture nutrient medium consists of inorganic salts (both micro- and
macro-elements), a carbon source (usually sucrose), vitamins (e.g. nicotinic acid, thiamine,
pyridoxine and myo-inositol), amino acids (e.g., arginine) and growth regulators (e.g. auxins,
cytokinins and gibbrellins). Other compounds like casein hydrolysate, coconut milk, malt
extract, yeast extract, tomato juice, etc. may be added
Fig. 1. Various steps involved in the regeneration of whole plants using tissue culture techniques.
(A) Citrus plant with fruits, (B) Seedlings raised by germinating Citrus seeds on nutrient medium,
(C) Transfer of epicotyl explants onto shoot regeneration medium, (D) Induction of shoots from
explants, (E) Elongation of shoots, (F) Rooting of in vitro shoot, (G) Regenerated plant in a pot.
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UNIT
Fig. 2. Plant tissue culture room (A) and transgenic green-house (B).
for specific purposes. Plant hormones play a pivotal role in growth and differentiation of
cultured cells and tissues. An optimum pH (usually 5.7) is also very important.
There are two kinds of media - liquid and solid media, and in the later case a gelling agent such as
agar (a polysaccharide obtained from a red algae, Gelidium amansii) is added for solidification of
the medium. There are several types of media like MS, LS, B5 and Nitsch's medium, and the choice
of media is dictated by the plant species, variety or plant part. However, the most extensively
used nutrient medium is MS medium, which was developed by Murashige and Skoog in 1962. The
success of plant regeneration from cultured cells and tissues are largely governed by the
composition of the culture medium. The sterilization of nutrient media by autoclaving or
membrane filter-sterilization is utmost important to avoid microbial contamination.
Types of cultures
Organ culture: It deals with the culture of the isolated organs (like roots) under laboratory
conditions (in vitro), and different names are given depending upon the organ utilized for the
culture. For instance, the culture of roots, endosperm, ovary, ovule and anther are called as i)
Root culture, ii) Endosperm culture, iii) Ovary culture, iv) Ovule culture and v) Anther culture
respectively.
Explant culture: The culture of plant parts (explants) is known as explant culture. The explants
can be any part of the plant like the piece of stem, leaf, cotyledon, hypocotyls, etc. The explant
cultures are generally used to induce callus or plant regeneration.
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111
Callus culture: Callus refers to an unorganised mass of cells, which are generally
parenchymatous in nature (Fig. 3A). A variety of plant parts can be induced to show callusing
response, but the response varies with the composition of culture media. Generally, auxins are
added to culture medium for callus induction but the nature and quantity of auxin added depends
on the nature and source of explant and its genotype besides other factors. Callus cultures can be
maintained for prolonged period of time by repeated sub-culture. Callus cultures are used for
plant regeneration.
Cell suspension cultures: Single cells can be isolated from either callus or any other part of the
plant (e.g. leaf) and cultured in liquid medium. Both mechanical and enzymatic methods can be
used for isolation of plant cells. Mechanical method involves grinding of the tissue to a fine
suspension in a buffered medium followed by filtration/centrifugation to get rid of cell debris.
The enzymatic method is based on the usage of enzymes (pectinase/macerozyme), which
dissolve the middle lamella between the cells, i.e., the inter-cellular cement, to release single
cells. Once the cells have been isolated, they may be cultured by batch cultures or continuous
cultures, which have been discussed in detail (see the previous chapter). The cell suspension
cultures can be used for:
genetic transformation.
Mass cell culture: It involves the large-scale culture of cells in specially designed 'plant
bioreactors', which essentially do not have a stirrer (as plant cells are shear sensitive). In place of
stirrer, gas is gently bubbled, which provides stirring as well as meet the demand of a higher
oxygen supply. Mass cell cultures are ideal for producing pharmaceutically important secondary
metabolites.
Protoplast culture: Protoplasts are plant cells without cell wall (Fig. 3B) and can be isolated
from a variety of plant tissues (usually leaves, callus pieces, single cells or pollen grains) by
enzymatic method using cell wall digesting enzymes (cellulases, hemicellulases and pectinases).
Protoplasts are usually cultured by suspension culture in petri plates. As the protoplasts lack cell
wall, they can be utilized for many purposes such as:
Genetic transformation.
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UNIT
Protoplast fusion: The protoplasts from two different plant genotypes can be fused in the
presence of fusogenic agents like polyethylene glycol (PEG - most widely used and most
successful method for protoplast fusion) or by electro-fusion. The hybrid cells (heterokaryons)
can be selected by various methods such as the use of different antibiotic markers or fluorescent
dyes for two different protoplasts. Then, regenerated somatic hybrids from hybrid cells are
characterized by morphology and molecular analysis (e.g. RAPD). This technique offers a unique
method to raise new genotypes (i.e. somatic hybrids between distantly related plants or cybrids)
with useful agronomic traits.
Fig. 3. Callus culture of indica rice (A), protoplasts isolated from leaf mesophyll cells of tobacco
(B), multiple shoot regeneration from leaf explants of brinjal (C), regeneration of somatic
embryos from leaf explants of brinjal (D) and scanning electron microscopy photographs of the
various stages of somatic embryogenesis - globular (E), heart-shape (F) and torpedo (G) stage
embryos from the brinjal culture (D).
UNIT
113
Micropropagation
Vegetative propagation of plants is of considerable importance in agriculture, horticulture and
forestry as it provides the multiplication of uniform material for crop planting (Clones).
Traditionally, it is done by using cuttings, budding, grafting, corms, tubers and other vegetative
propagules. The main problem with this method is that it is labour-intensive, low productivity and
seasonal. Thus, tissue culture method of plant propagation, known as 'micropropagation' can be
used to overcome the problems mentioned above. This technique utilizes the culture of apical
shoots, axillary buds and meristems on suitable nutrient medium, and has been adopted for
commercialisation of important plants such as banana, apple, pears, strawberry, cardamom,
many ornamentals (e.g. Orchids) and other plants.
There are four defined steps in micropropagation method. These are:
Initiation of culture - from an explant like shoot tip on a suitable nutrient medium.
Shoot multiplication - multiple shoots formation (Fig. 4A) from the cultured explant.
Transplantation - the hardening of tissue culture raised plants (Fig. 4B) as they are
tender and subsequent transplantation to the green-houses or field.
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In recent years, much effort has been devoted to develop automated, robotised and more
efficient transplant production methods. In India, the Department of Biotechnology (DBT)
initiated a major network programme in 1989, and so far propagation protocols have been
developed for more than 60 elite plant species.
Virus-free plants
Most of the crops plants, particularly vegetatively propagated plants are systemically infected
with viruses. If the stock of a cultivar (vegetative propagules) are infected with a virus, the
entire clonal population raised with such stock will also be infected and reduce the yield and
quality significantly. Therefore, the production of virus-free plants is important to increase yield
and quality. Interestingly, the distribution of viruses in plants is uneven, and the apical or axillary
meristems are generally free from viral particles. This has enabled scientists to produce virusfree plants by culturing small meristems (usually less than 1 mm long) collected from virusinfected plants. Meristem culture technique is now successfully used to produce virus-free
plants (but not virus-resistant plants) in a number of important clonal plants like potato,
sugarcane, banana and apple. The scheme used for the production of virus-free plants by
meristem culture is essentially same as micropropagation technique, except that the starting
material (explant) is meristem which is dissected out from shoot tips of infected plants.
Artificial seeds
The artificial seeds (also called as synthetic seeds or somatic seeds) can be utilized for the rapid
and mass propagation of elite plant species as well as hybrid varieties. Artificial seeds are
produced either by encapsulating the somatic embryos in a protective coating, i.e., calcium
alginate beads or by desiccating the somatic embryos with or without coating. A typical artificial
seed is shown in Fig. 5.
Fig. 5. The diagrammatic representation of an artificial seed. The artificial seed coat (A),
somatic embryo at torpedo stage (B) and artificial endosperm (C).
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115
Embryo rescue
It is very difficult to produce hybrids in case of inter-specific and inter-generic crosses (crosses
between distantly related plants) because of abnormal development of the endosperm, which
causes premature death of the hybrid embryo and leads to sterile seeds. The embryo from such
sterile hybrid seeds can be excised at an appropriate time and cultured on a suitable nutrient
medium to produce novel hybrids which is otherwise not possible. This is known as embryo
rescue. Several useful hybrids were produced in a variety of crops using this technique.
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Fig. 6. Development of somatic hybrids in brinjal for transferring disease resistance from
wild species to the cultivated variety. Flowers and fruits of the cultivated
brinjal (A & B), somatic hybrids (C & D) and wild-type brinjal (E & F).
It is also possible to produce cytoplasmic hybrids (cybrids) through protoplast fusion in which the
genomes of one of the partners is lost. Alternatively, the isolated and purified organelles chloroplasts or mitochondria from one species can be fused with the recipient protoplasts from a
different plant species (known as organelle transfer or organelle uptake) to transfer useful
cytoplasmic traits like herbicide tolerance and cytoplasmic male sterility.
UNIT
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feeding by animals. However, many of the secondary products, especially various alkaloids are of
immense use in medicine (Table 1). Such compounds are produced in plants only in small
amounts, and therefore, they are quite expensive.
Table 1. Few examples of industrially important plant secondary metabolites produced
through cell and tissue cultures
Product
Plant source
Uses
Artemisin
Artemisia spp.
Antimalarial
Azadirachtin
Insecticidal
Berberine
Coptis japonica
Antibacterial,
Antiinflammatory
Capsaicin
Codeine
Papaver spp.
Analgesic
Digoxin
Digitalis lanata
Cardiac tonic
Diosgenin
Dioscorea deltoidea
Antifertility
Scopolamine
Datura stramonium
Antihypertensive
Quinine
Cinchona officinalis
Antimalarial
Shikonin
Lithospermum erythrorhizon
Taxol
Taxus spp.
Anticarcinogenic
Vincristine
Cathranthus roseus
Anticarcinogenic
The indiscriminative use of such valuable medicinal and other plants has brought them to near
extinction. A possible solution in this direction is provided by cell and root cultures. Certain
plant products such as shikonin are being produced commercially in large-scale using bioreactors.
Somaclonal variation
It has been observed that the long-term callus and cell suspension culture and plants regenerated
from such cultures are often associated with chromosomal variations (somaclonal variation). It is
this property of cultured cells that finds potential application in the crop improvement and in the
production of mutants (e.g., disease resistance in potato).
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Larkin and Scowcroft (1981) proposed the term 'somaclones' for plant variants obtained from
tissue cultures of somatic tissues. Similarly, if the tissue from which the variants have been
obtained is having gametophytic origin such as pollen or egg cell, it is known as 'gametoclonal
variation.
Cold storage - this uses the short- or medium-term storage of germplasm by using
shoot tip, nodal or meristem explant cultures. The storage in done under conditions
o
o
that impose slow growth such as low temperature (4 C or 15 C), nutrient limitation or
the addition of growth retardants (e.g. absisic acid) in medium.
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Fig. 7. Induction of crown gall on the wounded stem induced by wild-type virulent
Agrobacterium tumefaciens (A), and the schematic diagram showing the cloning of the gene
of interest in Ti-plasmid of Agrobacterium and its transfer to plant cells in culture
to produce transgenic plants with desirable traits (B).
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Fig. 8. The Bio-rad Gene gun machine, Model He1000 (A), and the schematic diagram
showing the bombardment of gold particles containing the DNA (gene of interest) on their surface
onto the plant cells in culture to produce transgenic plants with desirable traits (B).
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Transgene analysis
The selection of transformed plant cells from untransformed cells is an important step in the
plant genetic engineering. For selection, the gene of interest is transferred along with a genetic
marker gene (usually antibiotic resistant genes such as nptII that confer resistance to
kanamycin), so that only the transformed cells can be selected and regenerated on the selection
medium containing the selection agent (e.g. kanamycin). The untransformed cells are eliminated
on the selection medium as they are susceptible to the antibiotic (due to the absence of antibiotic
resistance gene). The segregation and stability of the transgene integration and expression in the
subsequent generations can be studied by genetic and molecular analyses (PCR, Southern,
Northern and Western blot analysis). (Refer to earlier chapters for details of these techniques).
King
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Stress tolerance
Crop plants are very productive under ideal cultural conditions, but ideal growing conditions
rarely occur. Moreover, plants encounter both biotic (viral, bacterial, fungal pathogens,
nematodes, insect pests and weeds) and abiotic (salinity, drought, extreme temperatures,
nutrient deficiency, etc.) stresses and these stresses cause a colossal loss of crop yield and
quality. The application of chemical and biological pesticides as well as the use of resistant
varieties is only partial success and these have certain limitations. Therefore, newer and
effective technologies are essential to meet the demand. In this context, biotechnological
strategies can be used to create transgenic plants with increased resistance to diseases and pests
as well as abiotic stresses.
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These are specific to particular group of insect pests, and are not harmful to useful insects like
butter flies and silk warms. Transgenic crops (e.g. cotton, rice, maize, potato, tomato, brinjal,
cauliflower, cabbage, etc.) with Bt genes have been developed for and such transgenic varieties
proved effective in controlling the insect pests and it has been claimed worldwide that it has led
to significant increase in yields, and dramatic reduction in pesticides use. Bt crops have already
been commercialised in several countries. The most notable example is Bt cotton (which contains
CryIAc gene) that is resistant to a notorious insect pest Bollworm and in the year 2002, Bt cotton
was adopted in India (Fig. 9B).
Disease resistance: Pathogens (viruses, fungi and bacteria) infect crop plants and drastically
reduce their yield and quality. Globally, several diseases have caused havoc at several points of
time in several countries. For instance, the great Irish famine resulted in great damage of potato
crop due to the late blight disease caused by fungal pathogen. India has also experienced a
famine in the form of Bengal famine due to destruction of rice caused by fungal pathogen. Thus,
there is a great concern about the control of plant diseases. Traditionally, farmers apply chemical
pesticides or use resistant crop varieties developed by the breeders, but these methods have
certain limitations.The alternative and useful strategy is the creation of disease resistant
transgenic crop plants by transfer of resistance genes from varied sources.
Virus resistance: There are several strategies for engineering plants for viral resistance, and
these utilizes the genes from virus itself (e.g. the viral coat protein gene). The virus-derived
resistance has given promising results in a number of crop plants such as tobacco, tomato, potato,
alfalfa and papaya. Some viral resistance transgenic plants like papaya resistant to papaya ring
spot virus (Fig. 9C) have been commercialised in some countries.
Fungi and bacteria: Plants respond to pathogens by inducing a variety of defence responses like
pathogenesis-related proteins (PR proteins), enzymes that degrade/destroy fungal cell wall,
antifungal proteins and compounds, phytoalexins, etc. Several transgenic crop plants showing
increased resistance to fungal pathogens are being raised with genes coding for the different
compounds mentioned above.
Some of these strategies, particularly the genes that encode for phytoalexins and cell wall
degrading enzymes were also used for producing bacterial resistant transgenic plants.
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Fig.9. Genetically engineered crops with desirable traits. Transgenic canola for herbicide (glyphosate)
tolerance before and after herbicide spray in the field (A); Bt cotton resistant to bollworm
(Helicoverpa armigera) with healthy bolls (inserted photo) and non-Bt cotton with infested
insect larvae (inserted photo) (B); Transgenic papaya resistant to papaya
ringspot virus (c).
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Male sterility
Male sterile plants are very important to prevent unnecessary pollination and to eliminate the
process of emasculation during the production of hybrid plants. These are created by introducing
a bacterial gene from Bacillus amyloliquefaciens that encode an enzyme barnase, which is an
RNA hydrolyzing enzyme that inhibits pollen formation if, the expression of this gene specifically
in the tapetal cells of anther using tapetal-specific promoter (e.g. TA29) to restrict its activity
only to the cells involved in pollen production. Male fertility can be restored by introducing
another gene from the same bacterium under the control of TA29, whose product barstar
(protein) tightly bind with RNase, so that the normal pollen are formed. This barnase/barstar
system was successfully utilized in producing male sterile/restorer lines in number of crops,
particularly mustard for hybrid production.
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Nutrient quality
Plants are the principle source of human nutrition. A small number of crop plants such as cereals,
legumes, vegetables and root crops supply most of the energy and nutrients (in the form of fats,
carbohydrates, proteins, vitamins and micronutrients) in the human diet. It is estimated that
about 800 million people, mostly in developing countries are malnourished (particularly children)
and suffering with several diseases due to the deficiency of nutrients. Therefore, the
improvement of nutritional quality of crop plants is extremely important. Transgenic crops with
improved nutritional quality have already been produced by introducing genes involved in the
metabolism of vitamins, minerals and amino acids. Few examples of genetic modification of
nutrition quality are described below.
Vitamin A: The source of vitamin A is either directly from animal food (e.g. milk, cheese and
meat) or indirectly from green leafy vegetables (e.g. carrots, spinach, tomatoes, and chillies)
and fruits (e.g. mango, melon and papaya), which contain carotenoids (beta-carotene - the
provitamin A) that are converted to vitamin A in the body. Vitamin A deficiency can lead to night
blindness, permanent blindness and skin disorder, among others. About 124 million children
worldwide are deficient in vitamin A and a quarter of a million go blind each year due to vitamin A
deficiency.
The staple food rice is extremely low in vitamin A, and therefore the improvement of vitamin A
content is very important. In a remarkable example of genetic engineering, Prof. Ingo Potrykus
and Dr. Peter Beyer developed genetically engineered rice (popularly known as 'Golden Rice'),
which is enriched in pro-vitamin A (beta-carotenoids) by introducing three genes involved in the
biosynthetic pathway for carotenoid under the control of endosperm-specific promoter, so that
gene products (enzymes) are synthesized in the rice endosperm. The seeds of Golden Rice are
yellow in colour because of pro-vitamin A is produced in the entire grain (Fig. 10A). Interestingly,
they have also further engineered this Golden Rice by introducing three more genes from
different organisms for iron source and its absorption. Similarly, transgenic crop plants are also
being produced to raise the level of other vitamins, particularly vitamin E (which is an essential
antioxidant in humans) and vitamin K (which is involved in bone formation).
Seed protein quality: The nutritional quality of cereals and legumes are limited because of
deficiency of the essential amino acids, i.e. lysine in cereals, and methionine and tryptophan in
pulses. Two genetic engineering approaches have been used to improve the seed protein quality.
In the first case, a transgene (e.g. gene for protein containing sulphur rich amino acids was
introduced into pea plant (which is deficient in methionine and cysteine, but rich in lysine) under
the control of seed-specific promoter. In the second approach, the endogenous genes are
modified, so as to increase the essential amino acids like lysine in the seed proteins of cereals.
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Edible vaccines
Crop plants offer a cost-effective bioreactor to express antigens which can be used as edible
vaccines. Some of the antigenic determinants on the surface of viruses and bacteria are proteins
made by the pathogen. The genes encoding these antigenic proteins can be isolated from the
pathogens and expressed in plants, and such transgenic plants or their tissues producing antigens
can be eaten for vaccination/immunisation (edible vaccines). The expression of such antigenic
proteins in crops like banana and tomato are useful for immunisation of humans since banana and
tomato fruits can be eaten raw. In case of animals, such genes can be expressed in crops like
alfalfa and other forage/fodder crops, which are suitable for vaccination. The edible vaccines
that are produced in transgenic plants have great advantages like the alleviation of storage
problems, easy delivery system by feeding and low cost as compared to the recombinant vaccines
produced by bacterial fermentation. Vaccinating people against dreadful diseases like cholera
and hepatitis B by feeding them banana/tomato, and vaccinating animals against important
diseases such as foot and mouth disease by feeding them sugar beets could be a reality in the near
future.
Biodegradable plastics
The biodegradable plastic, e.g. polyhydroxybutyrate (PHB) is being produced commercially by
fermentation with the bacterium Alcaligenes eutrophus. The main drawback of bacterial PHB is
its high production cost, making it substantially very expensive than synthetic plastics.
Alternatively, transgenic plants can be used as factories to produce PHB. The genetically
engineered Arabidopisis plants with the three genes involved in PHB synthesis from A. eutrophus
produced PHB globules exclusively in their chloroplasts without effecting plant growth and
development (Fig. 10B). The large-scale production of PHB may be easily achieved in tree plants
like populus, where PHB can be extracted from leaves. Industry has already begun to explore the
production of biodegradable plastics from transgenic plants.
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Fig. 10. Golden rice (A) and electron microscopic picture of chloroplast of transgenic Arabidopsis
showing PHB globules (B).
Other applications
There are many other applications of plant genetic engineering, which are listed below:
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129
Development of blue roses (Fig. 11A), which is otherwise not possible by conventional
plant breeding because of the absence of blue pigment in roses.
Development of blue coloured cotton (Fig. 11B) and also with other colours.
Undoubtedly, there will be many more exciting applications of plant genetic engineering in the
future.
Blue Rose
Fig. 12. Genetically modified blue rose (A) and blue coloured cotton (B).
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The safety of GM food for human and animal consumption (e.g. GM food may cause
allergenicity).
Transgenes may escape through pollen to related plant species (gene pollution) and
may lead to the development of super weeds.
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The GM crops may change the fundamental vegetable nature of plants as the genes
from animals (e.g. fish or mouse) are being introduced into crop plants.
The antibiotic resistance marker genes used to produce transgenic crops may
horizontally transfer into microbes and thus exacerbate problem of antibiotic
resistance in human and animal pathogens (i.e. transgenes may move from plants to
gut microflora of humans and animals).
Unfortunately, the public debate over the benefits and hazards of plant gene technology suffers
from an astounding array of misinformation, misunderstanding and manipulation. In fact, with
the continuing accumulation of evidence of safety and efficiency, and the complete absence of
any evidence of harm to the humans and animals as well as the environment, more and more
consumers are becoming comfortable because of the increased awareness about the potential of
the plant genetic engineering. Further, the transgenic crops (e.g. cotton, tomato, corn and
soybean) which have already entered the market place (which carry a label, i.e., GM crop or GM
food in several countries were subjected for the extensive field trials for environmental safety
related to wild species and competitive performance of transgenic and toxic effects as per the
standard regulatory policies before they were approved for commercialisation. In fact, in 2009,
a record of 14 million farmers from 25 countries cultivated 134 million hectares (330 million
acres) with the crops that were genetically engineered for herbicide, insect resistance, delayed
fruit ripening and improved oil quality. The global area of biotech crops planted from 1996 to 2009
is shown in Fig. 12.
UNIT
Fig. 12. Global area of biotech crops from 1996 to 2009 (in million hectares).
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Nevertheless, the thorough assessment of the risks associated with transgenics for plants,
animals and humans is important before they are released. Foods or food ingredients derived
from GMOs must be shown to be as safe as or safer than their traditional counterpart before they
can be recommended for public use. In general, many scientists believe that plant genetic
engineering is the 'technology' that can solve various human problems, especially hunger and
disease.
Review Questions
1.
What is meant by plant tissue culture? List the various steps involved in this technique?
2.
3.
Name a few useful secondary metabolites that can be produced through plant cell culture
technology.
4.
List the various applications of plant cell and tissue culture technology.
5.
Differentiate:
i)
ii)
6.
7.
8.
How are plant cells converted to protoplasts? Why are protoplasts more amenable to
produce somatic cell hybrids and cybrids?
9.
What is genetic engineering? Add a note on the various steps involved in this technology.
10.
11.
Explain how gene gun functions to deliver genes into plant cells?
12.
13.
What are the genetic engineering strategies to create the following traits in transgenic
crops:
14.
132
a)
Herbicide tolerance
b)
c)
Insect resistance
d)
Virus resistance
What is the common strategy to produce transgenic crops with delayed ripening and longer
shelf life of fruits?
UNIT
15.
What is meant by 'Golden Rice'? In what way it is different from the normal rice?
16.
Write briefly the benefits of biodegradable plastics that are produced from GM plants.
17.
18.
19.
Define:
Explant
Hairy roots
Clone
Roundup
Callus
PR proteins
Micropropagation
Bt genes
Encapsulation
Male sterility
Embryo rescue
Flavr Savr
Somatic hybrids
Molecular farming
Cybrids
PHB
Edible vaccines
T-DNA
Metabolic engineering
Crown galls
20.
ii)
iii)
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133
References
134
1.
Bhojwani SS & Razdan MK. 1996. Plant Tissue Culture: Theory and Practice (Revised
Edition), Elsevier, Amsterdam.
2.
Chrispeels MJ & Sadava DE. 2003. Plants, Genes and Crop Biotechnology (Second
Edition), Jones and Bartlett Publishers, Inc., Boston.
3.
Davey MR & Anthony P. 2010. Plant Cell Culture: Essential Methods, Wiley-Blackwell,
Oxford.
4.
Gamborg OL & Phillips GC.1996. Plant Cell, Tissue and Organ Culture, Narosa
Publishing House, New Delhi.
5.
Glick BR & Pasternak JJ. 1998. Molecular Biotechnology: Principles and Applications of
Recombinant DNA (Second Edition), ASM Press, Washington, D.C.
6.
Oksman-Caldentey K-M & Barz WH. 2002. Plant Biotechnology and Transgenic Plants,
Marcel Dekker, Inc., New York.
7.
8.
Slater A, Scott N & Fowler M. 2003. Plant Biotechnology: The Genetic Manipulation of
Plants, Oxford University Press, Oxford.
UNIT
CHAPTER
3
6.3.1. Introduction
Animal cells, just as plant cells, will continue to grow outside the living organism if supplied with
the appropriate nutrients and growth conditions. The process of growing of cells under laboratory
conditions is called Cell Culture. It is carried out in vitro ('within glass') as opposed to in vivo
('within the living'). The advantage and limitations of animal cell culture are given in Table 1. A
homogenous population of cells derived from a single parental cell is called a clone. Therefore,
all cells within a clonal population are genetically identical. The growth rate of animal cells is
relatively slow and usually require 18 to 24 hour to divide. This makes the animal cell culture
vulnerable to contamination, as a small number of bacteria would soon outgrow a larger
population of animal cells. The animal cell culture became a routine laboratory technique in
1950s after George Gay established the first human cell line (HeLa) from cervix cancer that led to
several important discoveries in medical sciences. The need for cell culture, especially at large
scales, became apparent with the need for viral vaccines.
Table 1. Advantages and limitations of animal cell culture.
Advantages
Disadvantages
Controlled physico-chemical
environment.
Scale-up is challenging.
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Advent of recombinant DNA technologies in the 1970s, enabled the expression of mammalian
genes in bacteria. However, soon it was realized that large proteins or glycoproteins of
therapeutic value could not be produced in bacteria. This prompted the usage of animal cell lines
for large-scale production of therapeutic proteins. The development of hybridoma technology
allowed us to produce large quantities of monoclonal antibodies of immense diagnostic and
therapeutic values. Further, recent advances in the area of stem cells, tissue engineering and
gene therapy are likely to open new treatment regimens.
Before you learn about the details of animal cell culture technology, it is instructive to know some
essential features of animal cell growth in culture.
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The characteristics of cells in culture usually depend on their original source within the animal.
Cells can be grown as adherent (anchorage-dependent) or suspension cultures (anchorageindependent). Adherent cells are usually derived from tissues of organs such as kidney where they
are not mobile and are embedded in connective tissue. They grow adhering to the cell culture
vessel. On the other hand, suspension cells do not attach to the surface of the culture vessel.
Virtually all suspension cultures are derived from cells of the blood system. This is because, these
cells (e.g., lymphocytes) are also suspended in plasma in vivo. The drawbacks of primary culture
are that they are time consuming and require the use of live animals or fresh tissue. There can be
considerable variation from one preparation to another particularly if prepared by different
people. These difficulties can be overcome by the use of secondary cell cultures or cell lines.
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Temperature
The mammalian cells are grown in incubators maintained at 37C. This temperature is chosen
because it is the core body temperature of Homo sapiens. Further, it has been observed that most
cells derived from the warm blooded animals will grow at this temperature.
pH
The regulation of extra-cellular and intra-cellular pH is essential for the survival of mammalian
cells. The correct pH is not only important for maintaining the appropriate ion balance but also
for maintaining optimal function of cellular enzymes and for optimal binding of hormones and
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growth factors to cell surface receptors. Even transient changes in pH can alter cell metabolism
which can lead to cell death. Most media strive to achieve and maintain the pH between 7 and
7.4. The regulation of pH is done using a variety of buffering systems. Most media use the
bicarbonate - CO2 buffering system. The interaction of CO2 derived from cells or atmosphere with
water leads to a drop in pH described by the equation below:
H2O + CO2
D
H2CO3
D
H+ + HCO3-
The bicarbonate content of the medium neutralizes the effect of increased CO2 according to the
+
Osmolality
The osmolality of the culture medium also has a significant bearing on cell growth and function. It
preserves the membrane integrity of cells. If the outside osmotic pressure becomes higher or
lower than that which must be maintained inside the cell, it will shrink or swell accordingly. The
osmolality of the medium used is determined by the media formulation. Salt and glucose are the
major contributors to the osmolality of the medium, although amino acids may also contribute
significantly. Almost all commercial media are formulated to have a final osmolality of around 300
mOsm. Osmolality can be checked directly with an osmometer.
Medium
The most commonly varied factor in culture systems is the growth medium. Medium is a mixture
of inorganic salts and other nutrients capable of sustaining cell survival in vitro. Having the
correct nutrient mixture can often be the determining factor in failure or success in cell culture.
The medium provides essential nutrients that are incorporated into dividing cells, such as, amino
acids, fatty acids, sugars, ions, trace elements, vitamins, cofactors, and ions necessary to
maintain the proper chemical environment for the cell. Some components may perform both
roles; for example, the sodium bicarbonate may be used as a carbonate source but also may play
an important role in maintaining the appropriate pH and osmolality. All media contain an energy
source, usually glucose. Many of the media contain phenol red as a pH indicator. This is very
helpful in monitoring the pH of the culture medium in an incubator. Highly acidic conditions turn
the phenol red into yellow while highly alkaline conditions turns the phenol red into pink color.
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139
bovine serum (FBS). Serum is also a source of various amino acids, hormones, lipids, vitamins,
polyamines and salts containing ions such as calcium, chloride, ferrous, ferric, potassium etc.
Current practice is to minimize using blood-based supplements and switch to serum-free
medium due to some complications of FBS usage. Although not required for cell growth,
antibiotics such as penicillin and streptomycin are often used in culture medium to control the
growth of bacterial and fungal contaminants.
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bacteria or fungi which will outgrow animal cells. The Laminar Air Flow (LAF) hoods allow the
work area to be free of such contamination. A LAF hood essentially performs two functions:
1.
Protects the tissue culture from the operator (by providing a sterile environment).
2.
Protects the operator from the tissue culture (from possible infection risk).
Depending on the nature of cells/tissue being handled (especially infective agents), the biology
safety cabinets are designated as Class I to class III. The LAF hoods have continuous displacement
of air that passes through a high efficiency particle air (HEPA) filter that removes particulates
from the air. The hoods are equipped with a short-wave UV light source that can be turned on for a
few minutes to sterilize the surfaces of the hood just before use.
CO2 Incubator
The CO2 incubator is designed to reproduce as closely as possible the environmental conditions of
the living cells. The essential functions of the incubator are to maintain, the sterility of the
chamber, a constant temperature, an atmosphere with a fixed level of CO2 and high relative
humidity. A pan of water is kept at all times in the incubator chamber to maintain high relative
humidity and prevent desiccation of the culture medium and maintain the correct osmolarity
(Fig. 3A). The animal cells are grown in an atmosphere of 5-10% CO2 because the medium used is
buffered with sodium bicarbonate/carbonic acid and the pH must be strictly maintained.
Centrifuge
For most cell culture only low-speed centrifuges are required (Fig. 3B). A gentle braking action
helps prevent disruption of the separated bands of cells. In most cases cells should be centrifuged
at 20C; nevertheless low operation temperature is useful to avoid exposing cells to uncontrolled
higher temperatures.
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Inverted Microscope
In tissue culture vessels, for example a petri dish, the cells are present at the bottom with the
culture medium above. The inverted microscope allows the cells at the bottom to be visualized
because the optical system is at the bottom with the light source on top (Fig. 3C). Observation of
cultures in this way will give an immediate idea of the health and growth of cells. Microscopes
should be kept covered to protect from dust and the lights turned down when not in use.
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is important to check the identity of any newly acquired cell line for species of origin, tissue of
origin and the maintenance of specific properties.
Once a cell line has been established, its karyotype has to be determined. This will confirm the
species of origin, at least for those cells karyotyped, and determine the extent of gross
chromosomal changes in the line. Karyotypes may vary from being near normal (i.e., the vast
majority of cells in the culture have normal karyotypes) to being aneuploid. While a normal
karyotype is desirable, the presence of an abnormal karyotype does not preclude using the cells
for in vitro studies, especially if it has been demonstrated that the cells retain the normal
function expected of them. If a normal karyotype is required (e.g., cell lines to be used to create
transgenic animals), then special care must be taken in handling the cells to minimize
chromosomal changes. The stability of the karyotype depends on the species from which the cell
lines was derived, the growth conditions used, the way in which the cells are sub-cultured and
whether or not the cells are frozen.
Revival
Frozen cells should be thawed as rapidly as possible by placing the vial containing them into a
37C water bath with moderate shaking. This is to minimize ice crystal formation, which may
damage cells. After thawing the cells can be transferred directly into a tissue culture vessel
containing suitable media for further growth.
Calcium phosphate
Here HEPES-buffered saline solution is mixed with a calcium chloride solution containing DNA for
transfection to form a fine precipitate of calcium phosphate with DNA. The suspension of the
precipitate is then added to the monolayer of cells. The cells take up the calcium-phosphate- DNA
complexes by endocytosis and express genes.
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Lipofection
In this case, gene is transferred with the help of tiny vesicles of bipolar phospholipids that fuse
with the cell membrane, releasing the DNA into the cytoplasm.
Microinjection
It is the most efficient method of gene delivery into cells. Here, DNA is directly injected into the
nucleus using a fine glass capillary under a microscope. However this method acquire a great
effort as each and every cell has to be injected individually.
Electroporation
In this method, cells are mixed with the DNA and placed in a small chamber with electrodes
connected to a specialized power supply. A brief electric pulse is applied, which is thought to
'punch holes' in the cell membrane, enabling the cell to take up DNA.
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Roller bottles
In roller bottles, the cells adhere to the total curved surface area of the micro carrier beads,
thereby markedly increasing the available space for growth. These tissue culture bottles can be
used in specialized CO2 incubators with attachments that rotate the bottles along the long axis.
After each complete rotation of the bottle, the entire cell monolayer has transiently been
exposed to the medium. The volume of medium need only be sufficient to provide a shallow
covering over the monolayer (Fig. 5A).
Spinner cultures
Spinner cultures are used for scaling up the production of suspension cells. They consist of a flat
surface glass flask with a suspended central teflon paddle that turns and agitates the medium
when placed on a magnetic stirrer. Commercial versions incorporate one or more side arms for
sampling and/or decantation. The cells are not allowed to settle to the bottom of the flask and
thus cell crowding occurs only at very high densities. Stirring the medium improves gas exchange
(Fig. 5C).
Table:2
Proteins
Therapeutic use
Erythropoietin (EPO)
CHO cells
Anemia
Factor VIII
CHO cells
Hemophilia A
Factor IX
CHO cells
Hemophilia B
CHO cells
Infertility
CHO cells
GH deficiency
Interleukin 2 (IL2)
CHO cells
Cancer therapy
CHO cells
Stroke
Hybridoma cells
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Autoimmune diseases
145
Erythropoietin
Erythropoietin (EPO) is a glycoprotein hormone that is involved in RBC production
(erythropoiesis) and wound healing. EPO stimulates the bone marrow to produce more red cells
and thereby increase the oxygen-carrying capacity of the blood. It is produced in response to
hypoxia (shortage of oxygen) or anoxia (lack of oxygen) caused by anaemia. EPO is useful in the
treatment of certain types of anemia such as anemia due to cancer, chronic renal failure and
treatment of AIDS. Recombinant human EPO (r-HuEPO) has been produced using Chinese Hamster
Ovary (CHO) cell lines. The use of r-HuEPO is advantageous over blood transfusion as it does not
require donors or transfusion facilities, and there is no risk of transfusion-associated disease.
Factor VIII
Haemophilia A is a common heritable genetic disorder where the body lacks the ability to produce
Factor VIII required for blood clotting. Like EPO, factor VIII is also a glycoprotein and has been
produced in CHO cells due to its large structure.
Factor IX
Hemophilia B or Christmas disease is the second most common type of bleeding disorder due to
deficiency of factor IX. Recombinant Factor IX produced in CHO cells is used to treat
haemophilia B.
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147
Donor
Liver graft
Portal vein
Recipient
LT
Extraction of lymphocytes
from liver perfusate
IV infusion of
activated lymphocytes
3 days after LT
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must maintain an adequate number of cells (stem cells) that retain the potential to proliferate to
make good such losses. The most well studied process has been the formation of blood cells
(haematopoiesis). It was known in case of mouse that haematopoiesis occurs in the spleen and
bone marrow. In human being about 100,000 haematopoietic stem cells produce one billion RBC,
one billion platelets, one million T cells, one million B cells per Kg body weight per day. The field
of stem cell research was established in 1960s by Ernest McCulloch and James Till at the
University of Toronto.
The two broad types of mammalian stem cells are: embryonic stem (ES) cells that are isolated
from the inner cell mass of blastocysts, and adult stem cells that are found in adult tissues. The
ES cells are pleuripotent and can differentiate into all types of specialized tissues (Fig. 9). The
adult stem cells are multipotent (lineage restricted) and act as a repair system for the body by
maintaining the normal turnover of regenerative organs, such as, blood, skin, or intestinal tissues
(Fig. 10). Stem cells are now routinely grown and transformed into specialized cells such as,
muscles or nerves through cell culture and used in medical therapies. The stem cells are useful in
many medical conditions where cells are either dead or injured or abnormal, such as:
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149
Fig. 10. Cultivation of adult stem cells from bone marrow and their
differentiation into specialized cells.
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UNIT
retain the characteristics of founder cells, even after prolonged culture and extensive
manipulation.
b.
c.
could be used to create chimeric mice by taking ES cells from a black mouse and
implant it into the embryo of an albino mouse (white). The progeny so developed had
skin color of black and white (a chimera Fig. 11).
d.
e.
Now it is possible to selectively remove a gene (gene knock outs) and make other precise genetic
modifications in the mouse ES cells and create mouse models of human diseases. Such mouse
models have been extremely useful not only in understanding the genetic basis of a disease but
also in search for new diagnostic and therapeutic modalities.
In 1998, James Thomson developed a technique to isolate and grow human ES cells in culture.
The human ES cells can be derived from the inner cell mass of blastocyst or from human germ cells
before they initiate meiosis and cultured in a petri dish. Specialised cells can be grown in the
presence of specific growth factors such as fibroblast growth factor and platelet-derived growth
factor. The human ES cells have opened new possibilities for stem cell therapy in clinics.
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151
Review Questions
152
1.
Name two important products from Animal Cell Culture Technology. What are their
functions?
2.
Write two important features of cultured animal cells. Differentiate between primary
and secondary cell cultures.
3.
What are cell lines? How the growth characteristics of cell lines determined?
UNIT
4.
What is the importance of pH while culturing animal cells? How is the pH maintained in
culture media?
5.
What are some of the characteristic features of normal and transformed cells?
6.
7.
8.
9.
10. How is erythropoietin produced by animal cell culture? Write down the procedure
involved.
11. What is the mode of action of tPA? How is it produced by animal cell culture
technology?
12. How are monoclonal antibodies different from polyclonal antibodies? Write one
therapeutic application of monoclonal antibody.
13. What are stem cells? Describe the application of embryonic stem cell technology.
14. What is gene knock out? How is this useful in generating genetic models of human
disease?
15. What is meant by tissue engineering? Discuss some important medical applications of
tissue engineering.
References
1.
Das HK. 2010. Text Book of Biotechnology - 4th ed., Wiley India Pvt Ltd., New Delhi.
2.
Ian Freshney R. 2010. Culture of Animal Cells: A Manual of Basic Techniques and
Specialized Applications - 6th ed., Wiley-Blackwell, New Jersey.
3.
Masters JRW. 2000. Animal Cell Culture. A Practical Approach - 3rd ed., Oxford
University Press, Oxford.
4.
Morgan SJ & Darling. 1993. Animal Cell Culture, BIOS Scientific Publishers, Oxford.
5.
Ratledge C & Kristiansen B. 2006. Basic Biotechnology - 3rd ed., Cambridge University
Press, Cambridge.
6.
Sinha BK & Kumar R. 2008. Principles of Animal Cell Culture, International Book
Distributing Co., Lucknow.
7.
Smith JE. 2009. Biotechnology - 5th Ed, Cambridge University Press, Cambridge.
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