Section H Section H Cloning VectorsCloning Vectors

DESIGN OF PLASMID VECTORS

BACTERIOPHAGE VECTORS

COSMIDS, YACs AND BACs

EUKARYOTIC VECTORS

Content

H1 Design of Plasmid Vectors

Fig. 1. (a) Screening by insertional inactivation of a resistance gene; (b) replica plating.

H1 Design of Plasmid Vectors

Fig. 2. (a) A plasmid vector designed for blue–white screening; (b) the colonies produced by blue–white screening.

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 Design of Plasmid Vectors

Fig. 3. A multiple cloning site at the 5′-end of lacZ′

Ampr

ori

pUC18(3 kb)

MCS (Multiple cloning sites,多克隆位点）

Lac promoter

lacZ’

H1-2 A plasmid vector for gene expression

Expression vectors: allowing the exogenous DNA to be inserted, stored and expressed.

1. Promoter and terminator for RNA transcription are required.

2. Intact ORF and ribosomal binding sites (RBS) are required for translation.

Expression vector (transcription and translation).

Promoters1. lacUV-5: a mutant lac promoter which is in

dependent of cAMP receptor protein.

2. lPL promoter

3. Phage T7 promoter

Fused proteins

Individual proteins

Fig. 4. A plasmid designed for expression of a gene using the T7 system

H2 Bacteriophage vector

Tow examples: H2-1 λ phage bacteriophageλ λ replacement vector H2-2 M13 phage M13 phage vector Cloning in M13 Hybrid plasmid-M13 vectors

.viruses that can infect bacteria.

.48.5 kb in length

.Linear or circular genome (cos ends)

λ phage

Lytic phase (Replicate and release)

Lysogenic phase (integrate into host genome)Fig. 1. (a) Phage λ and its genome;

(b) the phage λ cos ends.

λ replacement vector

. Replace the nonessential region of the phage genome with exogenous DNA

. high transformation efficiency (1000-time higher than plasmid)

Pro

tein

coat

Fig. 2. Cloning in a λ replacement vector.

Plaques: the clear areas within the lawn where lysis and re-infection have prevented the cells from growing.

Recombinant l DNA may be purified from phage particles from plaques or from liquid culture.

H2-2 M13 phage vector

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.

M13 mp18 vector

H3 COSMIDS, YACs AND BACs

. Cloning large DNA fragments

. Cosmid vectors

. YAC vectors

. Selection in S. cerevisiae

. BAC vector

Analysis of eukaryotic genes and genome organization of eukaryotic requires vevtors with a larger capacity for cloned DNA than plasmids or phage λ.

H3-1 Cloning large DNA fragments

(Eukaryotic Genome project)

H3-2 Cosmid vectors

Cosmids use the λ packaging system to package large DNA fragments bounded by λ cos sites, which circularize and replicate as plasmids after infection of E.coli cells. Some cosmid vectors have two cos sites, and are cleaved to produce two cos ends, which are ligated to the ends of target fragments and packaged into λ particles. Cosmids have a capacity for cloned DNA of 30-45 kb.

Formation of a cosmid clone

Fig. 1. Formation of a cosmid clone.

Yeast artifical chromosomes can be constructed by ligating the components required for replication and segreation of natural yeast chromosomes to very large fragments of target DNA, which may be more than 1 Mb in length.

Yeast artifical chromosome(YAC) vectors contain two telomeric sequences(TEL), one centromere(CEN), one autonomously replicating sequence(ARS) and genes which can act as selectable markers in yeast.

H3-3 YAC vectors

Selection for the presence of YACs of other vectors in yeast is achived by complementation of a mutant strain unable to produce an essential metabolite, with the correct copy of the mutant gene carried on the vector.

H3-4 Selection in S.cerevisiae

H4 Eukaryotic Vectors

1. Shuttle vectors

2. Yeast episomal plasmids (Yeasts)

3. Agrobacterium tumefaciens Ti plasmid (Plant

s)

4. Baculovirus (Insects)

5. Mammalian viral vectors (Mammalian)

Shuttle vectors

H4-1 Yeast episomal plasmids (YEps)

Vectors for the cloning and expression of genes in Saccharomyces cerevisiae.

Replicate as plasmid from 2m origin

integrate by recombinantion

YEp vector

H4-2 Agrobacterium tumefaciens Ti plasmid

crown gall or tumor

Plant gene engineering using T-DNA vector

H4-3 Baculovirusbaculovirus is an insect virus which is used for the overexpression of animal proteins in insect cell culture.

H4-4 Mammalian viral vectors

Fig 1. Gene expression by SV40. Early genes are in red, late genes are in green. Note: - - - - indicates regions of the primary transcript which are removed in the alternatively processed mRNA. Cross-hatched area indicates region of RNA translated in different reading frames according to which alternatively spliced transcript is being translated Modified from Fiers et al.,Nature 273:113

Fig 2. retrovirus lifecycle

Gene transfer

Genes may be introduced into plant of animal cultured cells without the use of a special eukaryotic vector.

Bacterial plasnids carrying eukaryotic genes may remain transiently in cells without replication or may integrate into the host genome by recombination at low frequency.