Molecular Biology Course. Section H Cloning Vectors

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1 Molecular Biology Course Section H Cloning Vectors

2 Cloning vectors H1 Plasmid vecters H2 Bacteriophage vectors H3 Cosmids and YACs H4 Eukaryotic vectors

3 Cloning vectors H1 Design of Plasmid Vectors H1-1 A plasmid vector for cloning H1-2 A plasmid vector for gene expression

4 H1 Design of Plasmid Vectors H1-1 A plasmid vector for cloning 1. autonomously replicating independent of host s genome. 2. Easily to be isolated from the host cell. (Plasmid preparation) 3. Selective markers: Selection of cells (1) Containing vector: one marker is enough (2) Containing ligation products: # twin antibiotic resistance # blue-white screening 4. Contains a multiple cloning site (MCS)

5 H1 Design of Plasmid Vectors Replica plating( 影印平板培养 ): transfer of the colonies from one plate to another using absorbent pad( 垫子 ) or Velvet ( 绒布 ). transfer of colonies +ampicillin these colonies have bacteria with recombinant plasmid + ampicillin + tetracycline ( 四环素 )

6 H1 Design of Plasmid Vectors

7 H1 Design of Plasmid Vectors lacz encode enzyme b-galactosidase IPTG 异丙基 -b-d- 硫代半乳糖苷 (Controlled by lac promoter) 诱导 X-gal (substrate of the enzyme) Blue product 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

8 H1 Design of Plasmid Vectors Recreated vector: blue transformants Recombinant plasmid: containing inserted DNA: white transformants Recreated vector (no insert) Recombinant plasmid (contain insert)

9 H1 Design of Plasmid Vectors 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.

10 H1 Design of Plasmid Vectors Some cloning vector can be used to transcribe the inserted DNA. (puc vectors)? Special transcriptional vectors: pgem series containing promoters from bacteriophages T7 and SP6 for in vitro transcription with the corresponding polymerases. Expression vector (transcription & translation).

11 H1 Design of Plasmid Vectors Expression vector (transcription & translation). Promoters 1. lacuv-5: a mutant lac promoter which is independent of camp receptor protein. (constitutive expression) 2. lp L promoter 3. Phage T7 promoter

12 H1 Design of Plasmid Vectors T7 promoter RBS Start codon MCS Amp r T7 expression vector Transcription terminator ori

13 Cloning vectors 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

14 H2 Bacteriophage vector λ phage viruses that can infect bacteria kb in length Linear or circular genome (cos ends) ( 裂解期 ) Lytic phase (Replicate and release) ( 溶原期 ) Lysogenic phase (integrate into host genome)

15 H2 Bacteriophage vector The phage λ cos ends 5 -CGGGGCGGCGACCTCG-3 3 -GCCCCGCCGCTGGAGC-5 Circular form Cleavage (during packaging) Ligation (after infection) GGGCGGGCGACCTCG-3 5 -CG + GC-5 3 -GCCCCGCCGCTGGA Linear form

16 H2 Bacteriophage vector λ phage

17 H2 Bacteriophage vector λ replacement vector Replace the nonessential region of the phage genome with exogenous DNA (~ 20 kb) high transformation efficiency (1000-time higher than plasmid)

18 H2 Bacteriophage vector Cloning inλ replacement vector( 置换载体 ) 1. Ligation 2. Packing with a mixture of the phage coat proteins ( 噬菌体外壳蛋白 ) and phage DNAprocessing enzymes( 噬菌体加工酶 ) 3. Infection and formation of plaques

19 H2 Bacteriophage vector Plaques: the clear areas within the lawn( 菌苔 ) where lysis and re-infection have prevented the cells from growing. Lawn of E.coli Clear plaque caused by l infection and lysis Recombinant l DNA may be purified from phage particles from plaques or from liquid culture.

20 H2 Bacteriophage vector l lysogens( 溶原体 ) in cloning techniques Genes or foreign sequences may be incorporated essentially permanently into the genome of E.coli by integration of a l vector containing the sequence of interest.

21 H2 Bacteriophage vector H2-2 M13 phage A filamentous phage Phage particles contain a 6.7kb circular single strand of DNA. After infection of a sensitive E.coli host,the complementary strand is synthesized,and the DNA replicated as a double-stranded circle,the replicative form( 复制型,RF) with about 100 copies per cell. Contrasting to phage l,the cell infected by M13 are not lysed, but continue to grow slowly, and single-stranded forms are continuously packaged and released from the cells as new phage particles.

22 H2 Bacteriophage vector Useful properties of M13 as a vector are RF 1. Replication form (RF, dsdna) of M13 phage can be purified and manipulated like a plasmid. 2. Phage particles (ssdna): DNA can be isolated in a single-stranded form ssdna has a number of applications, such as: DNA sequencing (Topic J2) Site-directed mutagenesis (Topic J5) Cloning into RF transfection (recombinant DNA) growth (plating on a cell lawn) plaques formation (slow growth)

23 Blue-white selection

24 H2 Bacteriophage vector Hybrid plasmid-m13 vectors Many small plasmid vectors (e.g. pbluescript) have be developed to incorporate M13 functionality ( 组合有 M13 功能的载体 ) Contain both the plasmid and M13 origin of replication Normally propagate as true plasmids Can be induced to form single-stranded phage particles by infection of the host cell with a helper phage( 辅助噬菌体 ).

25 Cloning vectors H3 Cosmids and YACs H3-1 Cloning large DNA fragments (> 20 kb) H3-2 Cosmid vectors H3-3 YAC vectors H3-4 Selection in S. cerevisiae ( 啤酒酵母筛选 )

26 H3 Cosmids and YACs H3-1 Cloning large DNA fragments (Eukaryotic Genome project) Analysis of eukaryotic genes and the genome organization of eukaryotes requires vectors with a larger capacity for cloned DNA than plasmids or phage l. Human genome (3 x 10 9 bp): large genome and large gene demand vectors with a large size capacity.

27 H3 Cosmids and YACs H3-2 Cosmid vectors 1. Utilizing the properties of the l phage cos sites in a plasmid vector. 2. A combination of the plasmid vector and the cos site which allows the target DNA to be inserted into the l head. 3. The insert can be kb.

28 Formation of a cosmid clone Digestion Ligation C) Packaging and infect

29 H3 Cosmids and YACs B cos SmaI Cloning in a cosmid vector S B cos B S cos B cos B

30 H3 Cosmids and YACs H3-3YAC vectors Can accommodate genomic DNA fragments of more than 1 Mb, and can be used to clone the entire human genes. Essential components of YAC vectors : Centromers (CEN), telomeres (TEL) and autonomous replicating sequence (ARS) for proliferation in the host cell. amp r for selective amplification and markers such as TRP1 and URA3 for identifying cells containing the YAC vector in yeast cells. Recognition sites of restriction enzymes (e.g., EcoRI and BamHI)

31 H3 Cosmids and YACs CEN4 ARS TRP1 SnaB SUP4: insertion site for red-white selection Cloning in YAC vector TEL B B TEL Digest with BamHI/SnaI Ligate with blunt ends Transfect into yeast

32 H3 Cosmids and YACs H3-4 Selection in S.cerevisiae 1.S. cerevisiae selectable markers do not normally 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 unable to make a specific compound. 3. Example: TRP1 mutants can t make try( 色氨酸 ), and can only grow on media supplemented with try. The presence of a plasmid containing gene encoding try enables the cell to grow on media without try.

33 Cloning vectors H4 Eukaryotic Vectors H4-1 Yeast episomal plasmids ( 酵母附加型质粒 ) H4-2 Agrobacterium tumefaciens ( 根瘤农杆菌 ) Ti plasmid (Plants) H4-3 Baculovirus ( 杆状病毒 )(Insects) H4-4 Mammalian viral vectors (Mammalian)

34 H4 Eukaryotic Vectors Transfection: The take-up of DNA into eukaryotic cells 1. more problematic than bacterial transformation 2. Much lower efficiency in the progress 3. Transfection methods Electroporation Microinjection liposome

35 H4 Eukaryotic Vectors Shuttle vectors 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. Most of the eukaryotic vectors are constructed as shuttle vectors

36 H4 Eukaryotic Vectors H4-1 Yeast episomal plasmids (YEps) Vectors for the cloning and expression of genes in S. cerevisiae based on 2m plasmid vector. 1. Based on 2 micron (2mm) plasmid which is 6 kb in length. One origin Two genes involved in replication A site-specific recombination protein FLP, which is homologous to l phage Int (l 噬菌体整合酶 ). 2. Normally replicate as plasmids, and may integrate into the yeast genome.

37 H4 Eukaryotic Vectors A YEp vector MCS Insert Figure 1

38 H4 Eukaryotic Vectors Replicate as plasmid from 2m origin YEp vector integrate by recombinantion

39 H4 Eukaryotic Vectors H4-2 Agrobacterium tumefaciens Ti plasmid Ti plasmid 200kb T-DNA plant chromosome Integrated T-DNA Gene induce crown gall ( 冠瘿瘤 )

40 H4 Eukaryotic Vectors crown gall or tumor

41 H4 Eukaryotic Vectors Recombinant Ti plasmid 1. Place the target gene in the T-DNA region of a Ti plasmid, then transform the recombinant Ti plasmid. 2. Recombinant T-DNA transformed into the A. tumefaciens cell carrying a modified Ti plasmid without T-DNA (T-DNA that are responsible for crown gall formation. The deleted T-DNA is called disarmed( 卸甲 ) T- DNA shuttle vector).

42 H4 Eukaryotic Vectors Plant gene engineering using T-DNA vector

43 H4 Eukaryotic Vectors H4-3 Baculovirus ( 杆状病毒 ) 1. Infects insect cells: Expressing polyhedrin protein( 多 角体蛋白 ) 2. The strong promoter can be used to overexpress foreign genes engineered. Thus, large quantities of proteins can be produced in infected insect cells. 3. Insect expression system is an important eukaryotic expression system.

44 H4 Eukaryotic Vectors H4-4 Mammalian viral vectors 1. SV40: 5.2 kb, can pack DNA fragment similar to phage l. 2. Retrovirus: single-stranded RNA genome, which copy to dsdna after infection. Have some strong promoters for gene expression Gene therapy

45 H4 Eukaryotic Vectors Gene transfer Genes may be transiently or permanently introduced into cultured eukaryotic cells without the use of vector in strict sense. Transient expression Integration