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

pUC18 3kb lacZ / ori amp r 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.

Amp r 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 amp r T7 promoter RBS （ ribosome binding site ） Star codon MCS Transcription terminater T7 expression vector 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 kb in length Linear 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 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. amp r 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 YEp vector Gene X Mutant LEU2 LEU2 Yeast genome ori amp r Integrate vector LEU2 Mutant LEU2 or H4-3 Yeast episomal plasmids

H4-4 Agrobacterium tumefaciens Ti plasmid crown gall or tumor

Plant genome Vir A Vir 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） VirAVirG VirB 、 C 、 D 、 E pp promoters Plant cellAgrobacteria ？

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