cloning vectors section h h1 design of plasmid vectors h2 bacteriophage vectors h3 cosmids h4 yac h5...

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