Transcript E. coli

Section G-I
Gene Manipulation
G1 DNA cloning: an overview
G2 Preparation of plasmid DNA
G3 Restriction enzymes
Section G: Gene manipulation
Yang Xu, College of Life Sciences
G1 DNA Cloning: An Overview
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DNA cloning
Sub-cloning
DNA libraries
Screening libraries
Section G: Gene manipulation
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Vectors
Plasmids
Hosts
Analysis of clone
Yang Xu, College of Life Sciences
DNA cloning
• Definition: It is the making process to place the target gene (the
gene of interest) in a vector (an autonomously replicating piece
of DNA), forming recombinant DNA, which then is placed into
another host species.
E
E S
E
Ampr
Ori
target gene
vector
Section G: Gene manipulation
Host
transformant
Yang Xu, College of Life Sciences
DNA cloning: Basic Process
Example: plasmid (vector)
and E. coli (host)
(1) Preparation of plasmid DNA containing the cloned target gene.
(2) Digestion of the plasmid with restriction endonucleases.
(3) Separation of the fragments by agarose gel electrophoresis.
(4) Purification of the desired target fragment.
(5) Ligation of the fragments, to form a new recombinant molecule.
(6) Transformation of the ligated plasmid into an E. coli strain.
(7) Selection of transformed bacteria (see Topic G4).
(8) Analysis of recombinant plasmids (see Topic G4).
Section G: Gene manipulation
Yang Xu, College of Life Sciences
Subcloning
• Definition: It is the process to
transfer of a fragment of cloned
DNA from one vector to another.
Experimental steps:
1. Preparation: of the plasmid
①
2. Digestion of the plasmid
3. Separation of the fragments
②
4. Purification of target fragment
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5. Ligation of the fragments
-+
6. Transformation
7. Selection of transformed
bacteria.
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8. Analysis of plasmids.
Section G: Gene manipulation
⑦
⑥
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⑧
Analysis
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DNA libraries
• Definition: DNA libraries are sets
of DNA clones (vectors/hosts),
each of which has been derived
from the insert of a different
fragment into a vector followed by
propagation in the host.
• Classification and features:
1. Genomic libraries:
• They are derived from random
fragments of DNA from the
genomes of species by shotgun
approach;
• The approach may be an
inefficient of finding a gene,
especially in eukaryotic genomes,
where much of the DNA is
noncoding.
Section G: Gene manipulation
Yang Xu, College of Life Sciences
DNA libraries
2. cDNA libraries:
(ProteinCellmRNAcDN
AVectorHost)
• They are derived from the
mRNA by reverse
transcription and are then
inserted into a vector;
• cDNA libraries are efficient
for finding and cloning a gene,
but only the coding region,
not the surrounding genomic
sequences.
Section G: Gene manipulation
Yang Xu, College of Life Sciences
Screening libraries
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Definition: It is the process to use a DNA probe to
find the clone that contains the gene interested.
• DNA probe: It is a radioactively labeled short DNA
sequence which is partially complementary to a
region of the target gene sequence, therefor, the
target gene or clone can be detected by its
hybridization.
• Making of the probes:
1. The probe sequence might be an oligo-nucleotide
derived from the sequence of the protein product of
the gene;
2. From a related gene from another species.
3. An increasingly important method for the
generation of probes is PCR
Section G: Gene manipulation
Yang Xu, College of Life Sciences
Vectors-I: Features of vectors
• The features of vectors:
– Vectors must normally be capable of being replicated
and isolated independently of the host's genome;
– Vectors also have a selectable marker, a gene which
allows host cells conferring resistance to a toxin.
– There are some vectors, for example phage  (see
Topic H2), which can incorporate DNA into the host
genome for longer term expression of cloned genes.
B
ampr
pBR322
tetA
Ori
Section G: Gene manipulation
Yang Xu, College of Life Sciences
Vectors-II: Types of vectors
• The common vectors:
1. Plasmids:
– Circular plasmid of E. coli: used in E. coli (host);
– Yeast episomal plasmids: used in yeast;
– Agrobacterium tumefaciens Ti plasmid: in plant.
2. Bacteriophages (viruses infecting bacteria; see Topic R2):
– Phage : also been used in E. coli, for cloning larger fragments
– Phage M13: used to clone ssDNA used in E. coli.
3. Cosmids (plasmid-bacteriophage hybrids): (see Topic H3).
4. Artificial chromosomes: for cloning huge fragments from humans.
– BAC: Bacterial artificial chromosomes (in E. coli);
– YAC: Yeast artificial chromosomes (in Yeast).
5. Virus: for other eukaryotic cells in culture
– SV40;
– Retroviruses. (see Topic H4).
Section G: Gene manipulation
Yang Xu, College of Life Sciences
Plasmids
• The first cloning vectors to be used, in the mid
1970s, were natural plasmids originally from E.
coli.
• Structure and features: Plasmids are
 small in size, from 2 to around 200 kb
 extrachromosomal circular molecules
 which exist in multiple copies (up to a few hundreds)
 within the host E. coli cells.
 They contain an origin of replication (ori), which
enables them to be replicated independently.
Section G: Gene manipulation
Yang Xu, College of Life Sciences
Plasmids
• Resistance gene: They usually carry a
few genes, one of which may confer
resistance to antibacterial substances:
1. The most widely known resistance gene
is ampr gene, which encodes the enzyme
-lactamase, that degrades penicillin
antibiotics such as ampicillin.
2. Another is the tetA gene, which encodes a
transmembrane pump able to remove the
antibiotic tetracycline from the cell.
Section G: Gene manipulation
Yang Xu, College of Life Sciences
Hosts
• The common hosts:
• E. coli: The initial isolation and analysis of DNA
fragments is almost always carried out using the E. coli
as the host (for circular plasmid, phage , phage M13,
cosmid, BAC);
• Yeast: It is being used to manipulate very large
fragments of the human genome (for episomal plasmid,
YAC).
• Other cells:
– Agrobacterium tumefaciens (for Ti plamid);
– Insect cells (for baculovirus);
– Eukaryotic cells (for SV40, retroviruses, shuttle
vectors).
Section G: Gene manipulation
Yang Xu, College of Life Sciences
Analysis of clone
• Once a clone containing a target gene is identified, the structure
of the cloned fragment may be investigated:
1. further using restriction mapping, the analysis of the
fragmentation of the DNA with restriction enzymes (see Topic J1)
and by agarose gel electrophoresis using marker of known sizes
(see Topic G3),
2. or ultimately by the sequencing of the entire fragment (see Topic
J2).
3. The sequence can then be analyzed by comparison with other
known sequences from data bases, and the complete sequence of
the protein product determined (see Topic J2).
Section G: Gene manipulation
Yang Xu, College of Life Sciences
G2 Preparation of Plasmid DNA
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Bacterial culture
Alkaline lyses
Phenol extraction
Ethanol precipitation
Cesium chloride gradient
Section G: Gene manipulation
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Plasmid mini-preparation
Harvest cells
Cells
By centrifugation
Extraction:
Phenol-chloroform
1. Resuspend cell pellet; 2. Add lysozym
Plasmid
RNA
Protein
3. Add detergent/NaOH; 4.Neutrize with
KOAc
Protein, chromosomal DNA
and membranes
CsCl gradient
Aqueous
(plasmid + RNA)
Protein
Plasmid
Denatured
protein
Phenol-chloroform
Chromosomal/
inear DNA
RNA
Take aqueous
1. Ethanol precipitate
2. RNase
Section G: Gene manipulation
Pure plasmid
1. Collect supercoiled
plasmid band
2. Ethanol precipitate
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G3 Restriction Enzymes and
Electrophoresis
• Digestion, separation and purification
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Restriction endonucleases
Restriction sequences
Cohesive ends
Restriction digests
Agarose gel electrophoresis
Section G: Gene manipulation
Yang Xu, College of Life Sciences
Digestion: Restriction endonucleases
• Function: Restriction-modification systems occur in many
bacterial species, and constitute a defense mechanism against
the foreign DNA into the cell.
• Structure: Restriction-modification system consist of two parts:
1. The first part is a restriction endonuclease, which recognizes a
short, symmetrical DNA sequence (Fig. 1), and hydrolyzes the
DNA backbone in each strand at a specific site.
2. The second part is a methylase, which adds a methyl group to a
C or A base within the same recognition sequences. This
modification protects the host DNA against the endonuclease.
Section G: Gene manipulation
Yang Xu, College of Life Sciences
Digestion: Restriction sequences
• Definition: It is a short palindromic
sequences, at which restriction
EcoR I
enzymes cleave DNA symmetrically
in both strands.
Recognizing
• Acting Steps: EcoRI as an example.
5’-GAATTC-3’
– Recognizing: The restriction
3’-CTTAAG-5’
endonucleases EcoR I acts as a
dimer, will only recognize a 6 bp Cutting
palindromic sequence.
5’-G-OH
P-AATTC-3’
HO-G-5’
– Cutting: The product of the cutting 3’-CTTAA-P
reaction is two restriction
annealing
fragments, each with a 5'-end with
5’-GAATTC-3’
a phosphate group and a 3'-end
3’-CTTAAG-5’
with a free hydroxyl group.
Section G: Gene manipulation
Yang Xu, College of Life Sciences
Digestion: Cohesive ends
• Definition: Some of the products of restriction
enzyme digestion have protruding ends, and these
ends are known as cohesive, or 'sticky' ends.
• Features: Those products of restriction enzyme
digestion with protruding ends have a further property:
– They can bind to any other end with the same overhanging
sequence, by base pairing (annealing) of the single-stranded
tails.
– For example, any fragment formed by an EcoR I cut can
anneal to any other fragment formed in the same way, and
may subsequently be joined covalently by ligation.
– In fact, in some cases, DNA ends formed by enzymes with
different recognition sequences may be compatible
Section G: Gene manipulation
Yang Xu, College of Life Sciences
Digestion: Restriction digests-I
• Application: Digestion of plasmid or genomic DNA is carried
out with restriction enzymes for analytical or cloning
preparation purposes.
• Examples: The digestion of a sample plasmid with two
different restriction enzymes, Bam HI and EcoR I.
E
E
E
Plasmid
E
X
with gene
E
E
X
B
B
E
EcoRI
BamHI
B
Section G: Gene manipulation
B
Yang Xu, College of Life Sciences
Digestion: Restriction digests-II
• Reaction system:
– All restriction enzymes require Mg2+ (magnesium)
usually at a concentration of up to 10 mM;
– but different enzymes require different :  pHs,  NaCl
concentrations or  other solution constituents.
• Reaction process: The DNA is  incubated at the  optimum
temperature (37C) with  the enzyme and  the appropriate
buffer, in a volume of perhaps 20 l.  A dye mixture is then
added to solution, and  the sample is loaded on to an agarose
gel.
Section G: Gene manipulation
Yang Xu, College of Life Sciences
That’s all for Section G-I
Section G: Gene manipulation
Yang Xu, College of Life Sciences