Vectors for Genetic Engineering

• A vector is a substance, usually a piece of DNA that carries a sequence of DNA or other
genetic material and introduces it into a new cell.
• Vectors act as vehicles to transfer genetic material from one cell to the other for
different purposes like multiplying, expressing, or isolation.
• Vectors are used as a tool in molecular cloning procedures so as to introduce the desired
DNA insert into a host cell.
• The DNA insert that is transmitted by a vector is termed recombinant DNA, and the
process is also known as recombinant DNA technology.
• Usually, the vectors are DNA sequences that carry different parts involved in different
functions. Vectors usually have an insert, also known as a transgene, that carries the
recombinant DNA and a larger sequence called the backbone of the vector responsible
for the structure of the vector.
• Vectors can be classified into different types depending on different characteristics. The
selection of vectors thus depends on the purpose of the process.
 Characteristics of a vector
• Vectors should be capable of replicating autonomously, which depends on the presence of particular
sequences in the vector that enables them to initiate replication and propagation within the host cell..
• The size of an ideal vector should also be small enough for it to be incorporated into the host genome. The
small size of the vector also enables it to incorporate a large-sized insert for transfer.
• Vectors should be easy to isolate and purify as these need to be recovered and reused for multiple
processes.
• For a vector to be effective, these should also have certain components that facilitate the process of
determining whether the host cell has received the vector. Most vectors used in this process have a gene that
either provides resistance to an antibiotic or produces a particular type of protein. These components are
called marker genes.
• Many vectors also require unique restriction enzyme recognition sites that enable the insertion of the vector
DNA in the presence of specific restriction enzymes. However, many vectors have been designed with a series
of restriction sites close to multiple cloning sites that increases the possible restriction enzymes that can be
used to digest the sequence.
• The introduction of vectors into the host cell should be easy, which depends on a number of factors.
• In the case of gene transfer processes, it is important that the vector is capable of integrating itself or the
recombinant DNA into the genome of the host cell.
• It is important that the introduction of recombinant DNA into the vector doesn’t affect the replication cycle of
the vector.
 Cloning Vectors
• Cloning vectors are vectors that are capable of replicating
autonomously and thus are used for the replication of the
recombinant DNA within the host cell.
• Cloning vectors are responsible for the determination of which host
cells are appropriate for replicating a particular DNA segment.
• Cloning vectors are of further different types that are defined by
different features unique to each type of vector.
Expression Vectors
• Expression vectors are vectors that enable the expression of cloned genes in order to
determine the successful cloning process.
• Usually, cloning vectors do not allow the expression of a cloned gene which is why the use of
expression vectors is required.
• The use of expression vectors facilitates the processing of introns in prokaryotes as these are
designed with restriction sites next to the regulatory region.
• The restriction sites on the vectors result in splicing of the cloned gene to permit the
expression of the gene under the regulatory system.
• The regulatory system in expression vectors consists of a promoter sequence, a termination
sequence, along a transcription termination sequence.
• The use of expression vectors is essential to determine the success of a cloning procedure and
the efficiency of selective markers on the vectors.
• Expression vectors can be plasmid-based or viral-based that are introduced into the host cells
in order to code for particular mRNAs.
• The expression vectors are often used for the production of proteins that can then be
visualized by different methods depending on the complexity of the host cell.
• Expression vectors are of varying degrees of complexity depending on whether they are to be
used in prokaryotic or eukaryotic cells.
Shuttle Vectors
• Shuttle vectors are that carry origins of replication from two different hosts, which enables them
to ‘shuttle’ between the two hosts.
• These vectors contain DNA plasmids that can usually replicate in both mammalian cells as well as
bacterial cells.
• Shuttle vectors function as hybrid vectors containing DNA sequences from bacterial plasmids and
mammalian viruses.
• The vectors contain three functional DNA sequences involved in the cloning process; a viral
replication origin, a bacterial replication origin, and a drug resistance gene.
• The presence of different replication sites and repair sequences enable the recovery and
maintenance of these vectors in bacterial cells.
• There are three different shuttle vectors depending on the type of replication system utilized by
the vectors.
• Transiently replicating shuttle vectors that need to be recognized by large T antigen in order to
replicate in human cells.
• Episomal shuttle vectors work to establish cell lines that can replicate permanently in the form of
plasmid DNA containing the DNA insert.
• Integrated shuttle vector undergoes replication only after fusion with particular cell types for
gene expression.
 Natural Plasmids-ColE1
• ColE1 is a plasmid found in bacteria. Its
name derives from the fact that it
carries a gene for colicin E1
(the cea gene).
• It also codes for immunity from this
product with the imm gene. In addition,
the plasmid has a series of mobility
(mob) genes.
• Its size and the presence of a single
EcoRI recognition site caused it to be
considered as a vector candidate.
pBR322
 One of the first widely used E. coli cloning vectors
 Created in 1977 in the laboratory of Herbert Boyer at the University of
California, San Francisco, it was named after Francisco Bolivar Zapata,
the postdoctoral researcher and Raymond L. Rodriguez.
 pBR322 is 4361 base pairs in length and has two antibiotic resistance
genes – the gene bla encoding the ampicillin resistance (AmpR) protein
and the gene tetA encoding the tetracycline resistance (TetR) protein.
 It contains the origin of replication of pMB1, and the rop gene, which
encodes a restrictor of plasmid copy number.
 The plasmid has unique restriction sites for more than forty restriction enzymes

 Eleven of these forty sites lie within the Tet R gene. There are two sites for

restriction enzymes HindIII and ClaI within the promoter of the TetR gene while

There are six key restriction sites inside the AmpR gene

 Promoters P1 and P3 are for the beta-lactamase gene. P3 is the natural promoter,

and P1 is artificially created by the ligation of two different DNA fragments to

create pBR322.

 P2 is in the same region as P1, but it is on the opposite strand and

initiates transcription in the direction of the tetracycline resistance gene.

 pUC19 is one of a series of plasmid cloning vectors created by Joachim Messing and co-workers in
1985.
 The designation "pUC" is derived from the classical "p" prefix (denoting "plasmid") and the
abbreviation for the University of California, where early work on the plasmid series had been
conducted
 It is a circular double stranded DNA and has 2686 base pairs.
 pUC19 is one of the most widely used vector molecules as the recombinants, or the cells into which
foreign DNA has been introduced, can be easily distinguished from the non-recombinants based on
color differences of colonies on growth media
 It has an N-terminal fragment of β-galactosidase (lacZ) gene of E. coli & The multiple cloning
site (MCS) region is split into codons 6-7 of the lacZ gene, providing for many restriction
endonucleases restriction site.
 pUC19 also encodes for an ampicillin resistance gene (ampR), via a β-lactamase enzyme that
functions by degrading ampicillin and reducing its toxicity to the host.
 pUC19 is small but has a high copy number which is a result of the lack of the rop gene.
 Insertion of foreign DNA into the MCS located within the lac Z gene causes insertional inactivation of
this gene at the N-terminal fragment of beta-galactosidase and abolishes intra-allelic
complementation. Thus bacteria carrying recombinant plasmids in the MCS cannot hydrolyse X-gal,
giving rise to white colonies, which can be distinguished on culture media from non-recombinant
cells, which are blue.
 Therefore, the media used should contain ampicillin, IPTG, and X-gal.
Blue White screen: The blue-white screen is a
screening technique that allows for the detection of
successful ligations in vector-based gene cloning.
DNA of interest is ligated into a vector. The vector is
then transformed into competent cell (bacteria). The
competent cells are grown in the presence of X-gal
(galactose). If the ligation was successful, the
bacterial colony will be white; if not, the colony will
be blue. This technique allows for the quick and easy
detection of successful ligation.
Phage vectors
•Bacteriophage vectors are viruses that only infect bacteria and transform them efficiently while
carrying large inserts.
•Bacteriophages or phages have higher transformation efficiencies which increase the chances of
recovering a clone containing the recombinant DNA segments.
•The most important feature of a phage is the packaging system which enables the incorporation of
large eukaryotic genes and their regulatory elements.
•The use of phages also facilitates the isolation of larger quantities of DNA that can be used for the
analysis of the insert.
•Even though there are a number of phages that can and have been used as vectors, phage λ is the
most convenient cloning vector.
•It can selectively package a chromosome about 50 kb in length, and the size of the phage can be
adjusted by removing the central part of the genome as it is not necessary for replication or the
packaging of the donor DNA.
•The use of a bacteriophage vector that can incorporate larger DNA segments decreases the number
of clones required to obtain a particular DNA library with the entire genome of the organism.
•Phage vectors are also effective as cloning vectors as the recombinant molecules formed after the
cloning process are packaged into infective particles that can then be stored or handle efficiently.
•Some of the common phages used as vectors include M13 phages, λ phages, and P1 phages.
What is a Cosmid?
A Cosmid, referred as a hybrid plasmid, comprises cos
sites extracted from Lambda phage particles and a
plasmid. These cos sites are long fragments of DNA with
200 basepairs. Hence, they have cohesive or sticky ends
that allow the plasmid to fit into the viral DNA.
Therefore, the cos sites are vital for packaging of the
DNA.
There are three cos sites;
•cosN site – involved in nicking the DNA strand by
terminase activity
•cosB site – engaged in holding the terminase.
•cosQ site – involved in preventing the degradation of the
DNA by DNases.
•Also, Cosmids can either replicate single-stranded DNA
or double-stranded DNA using a suitable origin of
replication. Cosmids usually contain antibiotic resistance
genes as markers for selection of transformants. Thus, the
use of a cosmid as a vector can facilitate the cloning, and
the restriction enzyme digestion of the vector can then
extract these fragments.
What is a Phagemid (Phasmid)?

• Phagemid, also termed as Phasmid, is a type of hybrid vector too. Phagemid contains a
special origin of replication termed as the f1 origin of replication. The f1 origin of
replication extracts from an f1 phage.
• Phagemid can replicate both single-stranded and double-stranded DNA.
• Replication can either take place as a plasmid while undergoing independent replication
or can get packaged into phage particles and finally infect bacterial host E coli.
• When infecting the E coli cells, the f1 phage requires the presence of a pilus. Hence, the
sex pili are important during in vitro packaging of Phagemids.
What are the Similarities Between Cosmid and Phagemid?
•Both Cosmid and Phagemid are cloning vectors used in recombinant DNA technology.
•Cosmid and Phagemid can replicate single-stranded and double-stranded DNA.
•Both can undergo independent replication similar to plasmids.
•Cosmid and Phagemid can undergo in vitro packaging and infect bacterial cells.
•Both Cosmid and Phagemid require a suitable origin of replication for cloning.
Cosmid vs Phagemid
What is the
Difference Cosmid is a hybrid vector that contains a cos site and a Phagemid is a plasmid that contains an F1 origin of
plasmid. replication of the F1 phage.
Between
Cosmid and Presence of Cos Sites
Phagemid?
Cos sites are present in cosmid and are necessary for in Cos sites are absent in phagemid.
vitro packaging.

Presence of F1 Origin of Replication

In cosmid, fi origin of replication may or may not present. F1 origin of replication is present in phagemid.

Presence of Antibiotic Resistant Genes

Antibiotic-resistant genes are present in cosmids to Antibiotic-resistant genes are absent in phagemids.
identify the transformants from non – transformants.

Requirement of In Vitro Packaging

Requires a cos site. Requires a sex pilus.
Artificial Chromosomes
• These are scaled-down versions of actual chromosomes that can
replicate themselves in the host cells, just like natural chromosomes do,
in a stable but non-integrating manner, to introduce large genes or
multiple genes into the mammalian or plant cell.
• They are used to introduce and control new DNA in a cell, to study how
chromosomes function, and to map genes in.
• Structurally, artificial chromosomes are DNA molecules of known
composition, synthesized by assembling known components to create a
chromosome that acts like a natural one.
• The chief advantage of an artificial chromosome is where researchers
need to accomplish a much larger piece of gene editing, such as when a
piece of DNA over 100 kb—encoding a whole biosynthetic pathway
containing numerous enzymes—needs to be added to a chromosome,
for instance.
The First Artificial Chromosome
• In 2014, geneticists first began to build an entire customized yeast
chromosome from the budding yeast Saccharomyces cerevisiae.
• They assembled a circular plasmid with a yeast centromere, a
replication origin, a marker gene, and two stretches of palindromic
telomere DNA, using recombinant DNA techniques and inserted it
into the yeast, where it became a simple linear molecule.
• These plasmids then replicated and segregated with each cell mitosis
cycle with over 99% accuracy, and were retained in dividing cultures
for at least 20 generations.
• By understanding how genes within a chromosome work together to
produce the essentials of life and normal metabolism, researchers
hoped that they could build plant and animal chromosomes, and even
human artificial chromosomes.
Yeast Artificial Chromosomes

• Artificial chromosomes for use in yeast and mammalian cells aim to

replicate these components on a single DNA molecule.

• In bakers yeast (Saccharomyces cerevisiae ), telomeres, centromeres,

and origins of replication have all been defined using genetics and have
been cloned.

• When assembled they can be grown as a small chromosome in bacteria

and form a vector capable of incorporating up to a million bases of
other DNA as a chromosome in yeast (a YAC).
Applications of YAC
• To investigate the properties of yeast chromosomes

• Most extensively used in the early phases of genome mapping

projects- preferred over Bacterial Artificial Chromosomes

• Useful for finding genes from information about the inheritance of

genetic diseases

• Useful for testing the function of genes in mice

 Bacterial Artificial Chromosomes
• A bacterial artificial chromosome (BAC) is an engineered DNA molecule
used to clone DNA sequences in bacterial cells (for example, E. coli).

• BACs are often used in connection with DNA sequencing. Segments of an

organism's DNA, ranging from 100,000 to about 300,000 base pairs, can
be inserted into BACs.

• The BACs, with their inserted DNA, are then taken up by bacterial cells.

• As the bacterial cells grow and divide, they amplify the BAC DNA, which
can then be isolated and used in sequencing DNA.
• A large piece of DNA can be engineered in a fashion that allows it be
propagated as a circular artificial chromosome in bacteria--so-called
bacterial artificial chromosome, or BAC.
• Each BAC is a DNA clone containing roughly 100 to 300 thousand base
pairs of cloned DNA.
• Because the BAC is much smaller than the endogenous bacterial
chromosome, it is straightforward to purify the BAC DNA away from
the rest of the bacteria cell's DNA, and thus have the cloned DNA in a
purified form.
• This and other powerful features of BACs have made them extremely
useful for mapping and sequencing mammalian genomes.
Mammalian Artificial Chromosomes
• Mammalian artificial chromosomes (MACs) are conceptually similar to YACs, but instead
of yeast sequences they contain mammalian or human ones.
• In this case the telomeric sequences are multimers (multiple copies) of the sequence
TTAGGG, and the commonly used centromeric sequence is composed of another repeated
DNA sequence found at the natural centromeres of human chromosomes and called
alphoid DNA.
• When added to suitable cell lines, these MAC DNAs form chromosomes that mimic those
in the cell, with accurate segregation and the normal complement of proteins at
telomeres and centromeres.
• Their primary use is not in genome mapping but as vectors for delivery of large fragments
of DNA to mammalian cells and to whole animals for expression of large genes or sets of
genes.
• They are still in development, and although gene expression has been demonstrated they
have not been used in a practical application.
Difficulties in creating mammalian
artificial chromosomes
• However, it is much more difficult to build mammalian artificial
chromosomes, for the same reasons that it was easier to build yeast
artificial chromosomes.
• These reasons include:
• Each of the 3 sequences for yeast chromosome function was well
characterized
• Efficient transformation and rapid growth of yeast permit speed, ease,
and control
• Yeast have small-sized chromosomes that are easier to assemble