Presentation on theme: "Section H Cloning Vectors"— Presentation transcript:
1 Section H Cloning Vectors Molecular Biology CourseSection HCloning Vectors
2 vectors Cloning vectors: to clone a gene in a vector IntroductionvectorsCloning vectors:to clone a gene in a vectorExpression vectors:allowing the exogenous DNA to be inserted, stored, and manipulated mainly at DNA levelIntegration vectors:allowing the exogenous DNA to be inserted, stored, and expressed.
4 H1 Design of Plasmid Vectors Cloning vectorsH1 Design of Plasmid VectorsH1-1 A plasmid vector for cloningH1-2 A plasmid vector for gene expression
5 H1-1 A plasmid vector for cloning H1 Design of Plasmid VectorsH1-1 A plasmid vector for cloningautonomously replicating independent of host’s genome.Easily to be isolated from the host cell. (Plasmid preparation)Selective markers: Selection of cells(1) Containing vector: one marker is enough (2) Containing ligation products:# twin antibiotic resistance# blue-white screening4. Contains a multiple cloning site (MCS)
6 Ampicillin resistant? yes yes Tetracycline resistant? No yes Screening by insertional inactivation of a resistance geneAmprTcroripBR322B X BBTcrAmprAmprXBorioriAmpicillin resistant? yes yesTetracycline resistant? No yes
7 H1 Design of Plasmid Vectors Replica plating: transfer of the colonies from one plate to another using absorbent pad or Velvet (绒布).transfer of colonies+ampicillin+ ampicillin+ tetracyclinethese colonies have bacteria with recombinant plasmid
9 Blue white screening H1 Design of Plasmid Vectors Lac promoter Ampr Screening by insertional inactivation of the lacZ geneLac promoterMCS (Multiple cloning sites,多科隆位点）AmprpUC18(3 kb)lacZ’oriThe 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.
10 H1 Design of Plasmid Vectors lacZ encode enzyme b-galactosidase lac promoter(substrate of the enzyme)X-galIPTGBlue productThe expression of active b-galactosidase has to be vector dependent for the selection purposelacZ’: 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
11 H1 Design of Plasmid Vectors Recreated vector: blue transformantsRecombinant plasmid: containing inserted DNA: white transformantsRecreated vector (no insert)Recombinant plasmid (contain insert)
12 Multiple cloning sites H1 Design of Plasmid VectorsMultiple cloning sitesMultiple restriction sites enable the convenient insertion of target DNA into a vectorAmproripUC18(3 kb)MCS (Multiple cloning sites,多科隆位点）Lac promoterlacZ’…ACGAATTCGAGCTCGGTACCCGGGGATCCTCTAGAGTCGACCTGCAGGCATGCA…. T h rA s n S er S e r Val Pro Gly Asp Pro Leu Glu Ser Thr Cys Arg His Ala Ser…EcoRISacIKpnISmaIXmaIBamHIXbaISalIHincIIAccIPstISphILac Z
13 H1-2 A plasmid vector for gene expression H1 Design of Plasmid VectorsH1-2 A plasmid vector for gene expressionExpression vectors: allowing the exogenous DNA to be inserted, stored and expressed.Promoter and terminator for RNA transcription are required.Intact ORF and ribosomal binding sites (RBS) are required for translation.
14 Some cloning vector can be used to transcribe a gene. (pUC vectors) H1 Design of Plasmid VectorsSome cloning vector can be used to transcribe a gene. (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).
15 H1 Design of Plasmid Vectors AmproripUC18(3 kb)MCS (Multiple cloning sites,多科隆位点）Lac promoterlacZ’The ORF of the inserted gene has to be in the same direction as that of the lacZA fusion protein contains the N-terminal sequence of lacZ and the inserted ORF will be produced
16 (transcription & translation). H1 Design of Plasmid VectorsExpression vector(transcription & translation).PromoterslacUV-5: a mutant lac promoter which is independent of cAMP receptor protein. (constitutive expression)lPL promoterPhage T7 promoterFused proteinsIndividual proteins
18 Fused proteins Lac fusions: (discussed) H1 Design of Plasmid VectorsFused proteinsLac fusions: (discussed)His-tag fusions: A sequence encodes His-tag was inserted at the N- terminus of the target ORF, which allows purification of the fusion protein to be purified by binding to Ni2+ column.
20 λ phage H2 Bacteriophage vector viruses that can infect bacteria. 48.5 kb in lengthLinear or circular genome (cos ends)Lytic phase (Replicate and release)Lysogenic phase (integrate into host genome)
21 H2-1λ phage H2 Bacteriophage vector cos cos Exogenous DNA (~20-23 kb) Protein coatDNALong (left)armshort (right)armcosNonessential regioncosExogenous DNA(~20-23 kb)
22 The phage λ cos ends H2 Bacteriophage vector 5‘-CGGGGCGGCGACCTCG-3’ 3’-GCCCCGCCGCTGGAGC-5’Cleavage Ligation(during packaging) (after infection)GGGCGGGCGACCTCG-3’5’-CG GC-5’3’-GCCCCGCCGCTGGACircular formLinear form
24 H2 Bacteriophage vector λ replacement vectorReplace the nonessential region of the phage genome with exogenous DNA (~ 20 kb)high transformation efficiency (1000-time higher than plasmid)
25 H2 Bacteriophage vector λ replacement vector2. Packing with a mixture of the phage coat proteins and phage DNA-processing enzymesLigation3. Infection and formation of plaques
26 H2 Bacteriophage 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.
27 lysogens in cloning techniques H2 Bacteriophage vectorlysogensin cloning techniquesGenes or foreign sequences may be incorporated essentially permanently into the genome of E.coli by integration of a vector containing the sequence of interest.
28 H2-2 M13 phage H2 Bacteriophage vector 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(RF) with about 100 copies per cell.Contrasting to phage ,the cell are not lysed by M13,but continue to grow slowly,and single-stranded forms are continuously packaged and released from the cells as new phage particles.
29 M13 phage vectors H2 Bacteriophage vector Replication form (RF, dsDNA) of M13 phage can be purified and manipulated like a plamid.Phage particles (ssDNA): DNA can be isolated in a single-stranded formDNA sequencing (Topic J2)Site-directed mutagenesis (Topic J5)Cloning (RF, like plasmid) transfection (recombinant DNA) growth (plating on a cell lawn) plaques formation (slow growth)
31 Hybrid plasmid-M13 vectors H2 Bacteriophage vectorHybrid plasmid-M13 vectorsSmall plasmid vectors (pBluescript) being developed to incorporate M13 functionalityContain both the plasmid and M13 origin of replicationNormally propagate as true plasmidsCan be induced to form single-stranded phage particles by infection of the host cell with a helper phage.
32 H3 Cosmids and YACs H3-1 Cloning large DNA fragments （> 20 kb) Cloning vectorsH3 Cosmids and YACsH3-1 Cloning large DNA fragments （> 20 kb)H3-2 Cosmid vectorsH3-3 YAC vectorsH3-4 Selection in S. cerevisiae (啤酒酵母）
33 H3-1 Cloning large DNA fragments H3 Cosmids and YACsH3-1 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 .Human genome (3 x 109 bp): large genome and large gene demand vectors with a large size capacity.
34 H3 Cosmids and YACsH3-2 Cosmid vectorsUtilizing the properties of the phage l 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.
35 Formation of a cosmid clone DigestionLigationC) Packaging and infect
36 Cloning in a cosmid vector H3 Cosmids and YACsBcosSmaICloning in a cosmid vectorSBBScoscosBcosB
37 H3-3YAC vectors H3 Cosmids and YACs Can accommodate genomic DNA fragments of more than 1 Mb, and can be used to clone the entire human genome, but not good in mapping and analysisEssential components of YAC vectors :Centromers (CEN), telomeres (TEL) and autonomous replicating sequence (ARS) for proliferation in the host cell.ampr 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)
39 Cloning in YAC vector H3 Cosmids and YACs SnaB CEN4 ARS TRP1 SUP4: insertion site for red-white selectionSUP4:CEN4ARSTRP1Cloning in YAC vectorTEL BB TELDigest with BamHI/SnaILigate with blunt endsTransfect into yeast
40 H3-4 Selection in S.cerevisiae H3 Cosmids and YACsH3-4 Selection in S.cerevisiaeSaccharomyces cerevisiae selectable markers do not confer resistance to toxic substancesGrowth 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.
42 Transfection: more problematic than bacterial transformation H4 Eukaryotic VectorsTransfection:The take-up of DNA into eukaryotic cellsmore problematic than bacterial transformationMuch lower efficiency in the progressTransfection methodsElectroporationMicroinjectionliposome
43 H4 Eukaryotic VectorsShuttle vectorsVectors 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
45 H4-1 Yeast episomal plasmids (YEps) H4 Eukaryotic VectorsH4-1 Yeast episomal plasmids (YEps)Vectors for the cloning and expression of genes in Saccharomyces cerevisiae.Based on 2 micron (2m) plasmid which is 6 kb in length.One originTwo genes involved in replicationA site-specific recombination protein FLP, homologous to l Int.2. Normally replicate as plasmids, and may integrate into the yeast genome.
50 Recombinant Ti plasmid H4 Eukaryotic VectorsRecombinant Ti plasmidPlace the target gene in the T-DNA region of a Ti plasmid, then transform the recombinant Ti plasmid. (WT is not good because of the crown gall formation)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 T-DNA shuttle vector).
51 H4 Eukaryotic VectorsPlant gene engineering using T-DNA vector
52 H4-3 Baculovirus Infects insect cells H4 Eukaryotic VectorsH4-3 BaculovirusInfects insect cellsThe strong promoter expressing polyhedrin protein can be used to over-express foreign genes engineered. Thus, large quantities of proteins can be produced in infected insect cells.Insect expression system is an important eukaryotic expression system.
53 H4-4 Mammalian viral vectors H4 Eukaryotic VectorsH4-4 Mammalian viral vectorsSV40: 5.2 kb, can pack DNA fragment similar to phage l.Retroviruss:single-stranded RNA genome, which copy to dsDNA after infection.Have some strong promoters for gene expressionGene therapy
54 H4 Eukaryotic VectorsGene transferGenes may be transiently or permanently introduced into cultured eukaryotic cells without the use of vector in strict sense.Transient expressionIntegration
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