The detailed steps of

synthetic biology
The rule of DNA expression
Choose a vector-plasmid
Site for the restriction enzyme-special
sequence
It can be cut by restriction enzyme

E.g. EcoRI, BamIII

It can cut and leave the sticky end
The target sequence
can insert in it.
Lac Z gene inactivated
X-gal (5-bromo-4-chloro-3-indolyl-β-d-
galactopyranoside) is an inexpensive
and sensitive substrate of β-
gal, whose hydrolysis results in
an intensely blue colored and
easily detectable end product,
5,5'-dibromo-4,4'-dichloro-
indigo.
A plasmid
Restriction enzyme
There are many common restriction enzyme sites that are found in plasmids, including:

1. EcoRI: Recognizes and cuts the sequence GAATTC

2. HindIII: Recognizes and cuts the sequence AAGCTT
3. BamHI: Recognizes and cuts the sequence GGATCC
4. XhoI: Recognizes and cuts the sequence CTCGAG
5. NotI: Recognizes and cuts the sequence GCGGCCGC

These restriction enzyme sites are commonly used in molecular biology research for cloning and
manipulating plasmids.
Antibiotics
There are several commonly used antibiotics for selection of E. coli in molecular biology experiments. These
include:
1. Ampicillin （氨苄青霉素） : It targets the cell wall synthesis of bacteria, and is widely used for selection in
cloning studies. The concentration of Ampicillin used is usually 50-100 µg/mL.
2. Kanamycin （卡那霉素） : This antibiotic targets protein synthesis, and is used at a concentration of 25-50
µg/mL for bacterial selection.
3. Chloramphenicol （氯霉素） : Targets protein synthesis, and is used at a concentration of 12.5-25 µg/mL for
bacterial selection.
4. Tetracycline （四环素） : It targets ribosome function in bacteria and is used at a concentration of 5-10 µg/mL.
5. Gentamicin （庆大霉素） : Targets protein synthesis and is commonly used for bacterial selection, usually at a
concentration of 10 µg/mL.
In general, the choice of antibiotic depends on the specific experimental needs and the antibiotic resistance profile
of the bacterial strain being used.
PCR-enzyme digestion-ligation method
In genetic engineering, it is generally performed in the following order: PCR amplification of the
required DNA sequence, followed by enzyme digestion of the plasmid, then ligation of the PCR
product and plasmid to obtain the recombinant plasmid. This order is commonly referred to as
the PCR-enzyme digestion-ligation method, which is currently one of the most commonly used
recombinant DNA techniques.
Gel Electrophoresis and Southern
Blotting
One indirect method of rapidly analyzing and
comparing genomes is gel electrophoresis
This technique uses a gel as a molecular sieve to
separate nucleic acids or proteins by size
A current is applied that causes charged molecules to
move through the gel
Molecules are sorted into “bands” by their size

Video: Biotechnology Lab

Fig. 20-9
TECHNIQUE

Mixture of Power
DNA mol- source
ecules of – Cathode Anode +
different
sizes

Gel
1

Power
source
– +
Longer
molecules

2 Shorter
molecules

RESULTS
Fig. 20-9a
TECHNIQUE

Mixture of Power
DNA mol- source
ecules of – Cathode Anode +
different
sizes

Gel
1

Power
source
– +
Longer
molecules

2 Shorter
molecules
Fig. 20-9b

RESULTS
In restriction fragment analysis, DNA fragments
produced by restriction enzyme digestion of a DNA
molecule are sorted by gel electrophoresis
Restriction fragment analysis is useful for comparing
two different DNA molecules, such as two alleles for a
gene
The procedure is also used to prepare pure samples of
individual fragments
Fig. 20-10

Normal -globin allele Normal Sickle-cell

allele allele

175 bp 201 bp Large fragment

DdeI DdeI DdeI DdeI Large

fragment
Sickle-cell mutant -globin allele

376 bp
376 bp Large fragment 201 bp
175 bp
DdeI DdeI DdeI

(a) DdeI restriction sites in normal and (b) Electrophoresis of restriction fragments
sickle-cell alleles of -globin gene from normal and sickle-cell alleles
A technique called Southern blotting combines gel
electrophoresis of DNA fragments with nucleic acid
hybridization
Specific DNA fragments can be identified by Southern
blotting, using labeled probes that hybridize to the
DNA immobilized on a “blot” of gel
Fig. 20-11
TECHNIQUE
Heavy
Restriction I II III weight
DNA + restriction enzyme fragments Nitrocellulose
membrane (blot)

Gel

Sponge

I Normal II Sickle-cell III Heterozygote Paper

-globin allele Alkaline
solution towels
allele
1 Preparation of restriction fragments 2 Gel electrophoresis 3 DNA transfer (blotting)

Radioactively labeled
probe for -globin gene

Probe base-pairs
I II III with fragments I II III

Fragment from
sickle-cell
-globin allele Film
over
Fragment from blot
normal -globin
Nitrocellulose blot allele
4 Hybridization with radioactive probe 5 Probe detection
Fig. 20-11a

TECHNIQUE
Heavy
Restriction I II III weight
DNA + restriction enzyme Nitrocellulose
fragments
membrane (blot)

Gel

Sponge
I Normal II Sickle-cell III Heterozygote Paper
-globin allele Alkaline
solution towels
allele
1 Preparation of restriction fragments 2 Gel electrophoresis 3 DNA transfer (blotting)
Fig. 20-11b

Radioactively labeled
probe for -globin gene

Probe base-pairs
I II III with fragments I II III

Fragment from
sickle-cell
-globin allele Film
over
Fragment from blot
normal -globin
Nitrocellulose blot allele
4 Hybridization with radioactive probe 5 Probe detection
DNA Sequencing
Relatively short DNA fragments can be sequenced by
the dideoxy chain termination method
Modified nucleotides called dideoxyribonucleotides
(ddNTP) attach to synthesized DNA strands of different
lengths
Each type of ddNTP is tagged with a distinct
fluorescent label that identifies the nucleotide at the
end of each DNA fragment
The DNA sequence can be read from the resulting
spectrogram
Fig. 20-12
TECHNIQUE

DNA Primer Deoxyribonucleotides Dideoxyribonucleotides

(template strand) (fluorescently tagged)

dATP ddATP
dCTP ddCTP
dTTP ddTTP
DNA
polymerase dGTP ddGTP

DNA (template Labeled strands

strand)

Shortest Longest

Direction
of movement Longest labeled strand
of strands

Detector

Laser
Shortest labeled strand
RESULTS Last base
of longest
labeled
strand
Last base
of shortest
labeled
strand
Fig. 20-12a

TECHNIQUE

DNA Primer Deoxyribonucleotides Dideoxyribonucleotides

(template strand) (fluorescently tagged)

dATP ddATP
dCTP ddCTP

DNA dTTP ddTTP

polymerase dGTP ddGTP
Fig. 20-12b
TECHNIQUE

DNA (template Labeled strands

strand)

Shortest Longest

Direction
of movement Longest labeled strand
of strands

Detector

Laser
Shortest labeled strand
RESULTS Last base
of longest
labeled
strand
Last base
of shortest
labeled
strand
Analyzing Gene Expression
Nucleic acid probes can hybridize with mRNAs
transcribed from a gene
Probes can be used to identify where or when a gene
is transcribed in an organism
Studying the Expression of Single Genes

Changes in the expression of a gene during embryonic

development can be tested using
◦ Northern blotting
◦ Reverse transcriptase-polymerase chain reaction

Both methods are used to compare mRNA from

different developmental stages
Northern blotting combines gel electrophoresis of
mRNA followed by hybridization with a probe on a
membrane
Identification of mRNA at a particular developmental
stage suggests protein function at that stage
Reverse transcriptase-polymerase chain reaction (RT-
PCR) is quicker and more sensitive
Reverse transcriptase is added to mRNA to make
cDNA, which serves as a template for PCR
amplification of the gene of interest
The products are run on a gel and the mRNA of
interest identified
Fig. 20-13
TECHNIQUE
1 cDNA synthesis mRNAs

cDNAs
Primers
2 PCR amplification

-globin
gene
3 Gel electrophoresis

RESULTS Embryonic stages

1 2 3 4 5 6
In situ hybridization uses fluorescent dyes attached to
probes to identify the location of specific mRNAs in
place in the intact organism
Fig. 20-14

50 µm
Studying the Expression of Interacting Groups of
Genes
Automation has allowed scientists to measure
expression of thousands of genes at one time using
DNA microarray assays
DNA microarray assays compare patterns of gene
expression in different tissues, at different times, or
under different conditions
Fig. 20-15
TECHNIQUE
Tissue sample
1 Isolate mRNA.

2 Make cDNA by reverse mRNA molecules

transcription, using
fluorescently labeled
nucleotides.

Labeled cDNA molecules

(single strands)
3 Apply the cDNA mixture to a DNA fragments
microarray, a different gene in representing
each spot. The cDNA hybridizes specific genes
with any complementary DNA on
the microarray.
DNA microarray

DNA microarray
4 Rinse off excess cDNA; scan
with 2,400
microarray for fluorescence.
human genes
Each fluorescent spot represents a
gene expressed in the tissue sample.