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Cloning Vectors
Section H
H1 Design of plasmid vectors
H2 Bacteriophage vectors
H3 Cosmids
H4 YAC
H5 Eukaryotic vectors
H1 Design of Plasmid Vectors
H1-1 A plasmid vector for cloning
H1-2 Twin antibiotic resistance
H1-3 Multiple cloning site
H1-4 Expression vectors
pUC183kb
lacZ/
ori
ampr
MCS
H1-1 A plasmid vector for cloning
1. Autonomously replicating independent of host’s genome.
2. Easily to be isolated from the host cell.
3. Selective markers: Selection of cells
twin antibiotic resistance
blue-white screening
4. Contains a multiple cloning site (MCS)
H1-2 Twin antibiotic resistance
A vector with two antibiotic resistance genes can be used to screen for recombinants if the target fragment is inserted into one of the genes, thus insertionally inactivating it.
Ampr
ori
pUC18(3 kb)
MCS
Lac promoter
lacZ’
The insertion of a DNA fragment interrupts the ORF of lacZ’ gene, resulting in non-functional gene product that can not digest its substrate x-gal.
H1-2 Blue white screening
lacZ encode enzyme b-galactosidase
lacZ’: a shortened derivative of lacZ, encoding N-terminal a-peptide of b-galactosidase.
Host strain for vectors containing lacZ’: contains a mutant gene encoding only the C-terminal portion of b-
galactosidase which can then complement the a-peptide to produce the active enzyme
IPTG
X-gal(substrate of the enzyme)lac promoter
Blue product
The expression of active b-galactosidase has to be vector dependent for the selection purpose
H1-3 Multiple cloning site
A multiple cloning site provides flexibility in choice of restriction enzyme or enzymes for colonies.
ori
ampr
T7 promoter
RBS ( ribosome binding site)Star codon
MCS
Transcription terminater
T7 expressionvector
Transcriptional vectors
Expression vectors
H1-4 Expression vectors
H2 Bacteriophage vector
H2-1 λ phage vectors
H2-2 M13 phage vectors
λ phage viruses that can infect bacteria. 48.5 kb in lengthLinear or circular genome (cos ends)
H2-1 λ phage vectors
5’-CG +3’-GCCCCGCCGCTGGA
GGGCGGCGACCTCG-3’ + GC-5’
5’-CGGGGCGGCGACCTCG-3’3’-GCCCCGCCGCTGGAGC-5’
Nonessential region
DNA
Long (left)arm
short (right)arm
Exogenous DNA(~20-23 kb)
λ replacement vector
Replace the nonessential region of the phage genome with exogenous DNA (~ 20 kb)
high transformation efficiency (1000-time higher than plasmid)
1. Replication form (RF, dsDNA) of M13 phage can be purified and manipulated like a plamid.
2. Phage particles (ssDNA): DNA can be isolated in a single-stranded form
• DNA sequencing • Site-directed mutagenesis
Cloning (RF, like plasmid) transfection (recombinant DNA) growth (plating on a cell lawn) plaques formation (slow growth)
H2-2 M13 phage vectors
H3 Cosmids and YACs
H3-1 Cosmid vectors
H3-2 YAC vectors
H3-3 Selection in S. cerevisiae
Cosmid vectors are so-called because
they utilize the properties of the phage
λ cos sites in a plasmid vector.
A combination of the plasmid vector
and the COS site which allows the
target DNA to be inserted into the l
head.
The insert can be 37-52 kb.
H3-1 Cosmid vectors
Essential components of YAC vectors :
1. Centromers (CEN), telomeres (TEL) and autonomous replicating sequence (ARS) for proliferation in the host cell.
2. ampr for selective amplification and markers such as TRP1 and URA3 for identifying cells containing the YAC vector in yeast cells.
3. Recognition sites of restriction enzymes (e.g., EcoRI and BamHI)
H3-2 YAC vectors
1.Saccharomyces cerevisiae selectable markers do not confer resistance to toxic substances
2.Growth of yeast on selective media lacking specific nutrients can serve for selection. Auxotrophic yeast mutants (营养缺欠型 ) are made as host strains for plasmids containing the genes complementary to the growth defect .
For example: TRP1 mutants can’t make tryptophan, and can only grow on media supplemented with tryptophan. The presence of a plasmid containing gene encoding tryptophane enables the cell to grow on media without tryptophan.
H3-3 Selection in S.cerevisiae
H4 Eukaryotic Vectors
H4-1 H4-1 Yeast episomal plasmids
H4-3 Shuttle vectors
H4-3 Yeast episomal plasmids
H4-4 Agrobacterium tumefaciens Ti plasmid
H4-5 Mammalian viral vectors
The take-up of DNA into eukaryotic cells.
H4-1 Transfection of eukaryotic cells
1. More problematic than bacterial transformation
2. Much lower efficiency in the progress
3. Transfection methods• Electroporation• Microinjection• liposome
Vectors contain sequences required for replication and selection in both E. coli and the desired host cells, so that the construction and many other manipulation of the recombinant plasmids can be completed in E. coli.
H4-2 Shuttle vectors
MCS
YEps —Vectors for the cloning
and expression of genes in
Saccharomyces cerevisiae.
DNA transport:transfection
protoplastes
calcium phosphate
electroporation
microinjection
2 origin
YEpvector
Gene X
Mutant LEU2
LEU2
Yeast genome
oriampr
Integrate vector
LEU2 Mutant LEU2
or
H4-3 Yeast episomal plasmids
H4-4 Agrobacterium tumefaciens Ti plasmid
crown gall or tumor
Plant genome
Vir AVir B
Vir C
Vir D
Vir E
T-region ( ~23kb)
Ti- plasmid~200kb
~40kb
植物组织损伤(酚类化合物)
VirA VirG VirB 、 C 、 D 、 Ep p promoters
Plant cell Agrobacteria
?
1. SV40: 5.2 kb, can pack DNA fragment similar to phage l.2. Retroviruss: • single-stranded RNA genome, which copy to dsDNA after infection.• Have some strong promoters for gene expression• Gene therapy
H4-5 Mammalian viral vectors