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Section H – Cloning vectors. H1 Design of plasmid vectors Ligation products, Twin antibiotic resistance, Blue-white screening, Multiple cloning sites,

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Presentation on theme: "Section H – Cloning vectors. H1 Design of plasmid vectors Ligation products, Twin antibiotic resistance, Blue-white screening, Multiple cloning sites,"— Presentation transcript:

1 Section H – Cloning vectors

2 H1 Design of plasmid vectors Ligation products, Twin antibiotic resistance, Blue-white screening, Multiple cloning sites, Transcription of cloned inserts, Expression vectorsLigation productsTwin antibiotic resistanceBlue-white screeningMultiple cloning sitesTranscription of cloned insertsExpression vectors H2 Bacteriophage vectors Bacteriophage λ, λReplacement vectors, Packaging and infection, Formation of plaques, λ Lysogens, M13 phage vectors, Cloning in M13, Hybrid plasmid-M13 vectorsBacteriophage λλReplacement vectorsPackaging and infectionFormation of plaques λ LysogensM13 phage vectorsCloning in M13Hybrid plasmid-M13 vectors H3 Cosmids, YACs and BACs Cloning large DNA fragments, Cosmid vectors, YAC vectors, Selection in S. cerevisiae, BAC vectorsCloning large DNA fragmentsCosmid vectorsYAC vectors Selection in S. cerevisiaeBAC vectors H4 Eukaryotic vectors Cloning in eukaryotes, Transfection of eukaryotic cells, Shuttle vectors, Yeast episomal plasmids, Agrobacterium tumefaciens TI plasmid, Baculovirus, Mammalian viral vectors, Direst gene transferCloning in eukaryotesTransfection of eukaryotic cells Shuttle vectorsYeast episomal plasmidsAgrobacterium tumefaciens TI plasmidBaculovirusMammalian viral vectorsDirest gene transferContents

3 H1 Design of plasmid vectors — Ligation products The most frequent unwanted product is recreated vector plasmid formed by circularization of the linear vector fragment

4 Minipreparations from a number of transformed colonies, and screening by digestion and agarose gel electrophoresis.

5 H1 Design of plasmid vectors — Twin antibiotic resistance Contain two antibiotic resistance genes : If a target DNA fragment is ligated into the coding region of one of the resistance genes the gene will become insertionally inactivated, and can be determined by the antibiotic resistance exhibited by the transformants.

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7 Screening by insertional inactivation of a resistance gene B X B B B X Amp r ori Amp r Tc r ori Amp r Tc r ori pBR322 B Recombinant Religated Ampicillin resistant? yes yes Tetracycline resistant? No yes

8 Replica plating: transfer of the colonies from one plate to another using absorbent pad or velvet transfer of colonies +ampicillin + tetracycline These colonies have bacteria with recombinant plasmid

9 H1 Design of plasmid vectors — Blue-white screening A more sophisticated procedure can be carried out on a single transformation plate; Blue white screening; Involves the insertional inavtivation of the gene lacZ.

10 Amp r ori pUC18 (3 kb) MCS (Multiple cloning sites ) Lac promoter lacZ’ Screening by insertional inactivation of the lacZ gene 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.

11 lacZ encode enzyme  -galactosidase IPTG X-gal (substrate of the enzyme) lac promoter Blue product i Operon RegulatoryGene p oz y a DNA m-RNA β -Galactosidase Permease Transacetylase Protein

12 Recreated vector: blue transformants Recombinant plasmid: white transformants (containing inserted DNA) Recreated vector (no insert) Recombinant plasmid (contain insert)

13 lacZ’: a shortened derivative of lacZ, encoding N-terminal a-peptide of b- galactosidase. Host strain carrying a mutant gene encoding only the C-terminal portion of  -galactosidase which can then complement the a-peptide to produce the active enzyme

14 H1 Design of plasmid vectors — Multiple cloning sites Multiple restriction sites enable the convenient insertion of target DNA into a vector Amp r ori pUC18 (3 kb) MCS Lac promoter lacZ’ …ACGAATTCGAGCTCGGTACCCGGGGATCCTCTAGAGTCGACCTGCAGGCATGCA…. Thr Asn Ser Ser Val Pro Gly Asp Pro Leu Glu Ser Thr Cys Arg His Ala Ser… EcoRISacIKpnI SmaI XmaI BamHI XbaI SalI HincII AccI PstISphI Lac Z’ Insertion of target DNA in MCS inactivates the lacZ’

15 H1 Design of plasmid vectors — Transcription of cloned inserts Some cloning vector : The pUC vectors have a promoter (lac) adjacent to the site of insertion of a cloned fragment, such a promoter could be used to transcribe the inserted DNA, either to produce an RNA transcript in vitro (used as a hybridization probe), or to express the protein product of a gene. Special transcriptional vectors The pBluescript Ⅱ SK has promoters from bacteriophages T7 and SP6 flanking an MCS.

16 H1 Design of plasmid vectors — Expression vectors (1)Promoter and terminator for RNA transcription are required. (2)Intact ORF and ribosomal binding sites are required for translation.

17 Strong Promoters Promoter : 1.lacUV-5: a strong mutant lac promoter independent of cAMP receptor protein (CRP or CAP). 2.lP L promoter 3.Phage T7 promoter Fusion protein and fusion tag 1.Defined epitope : a small piece of peptide sequence containing a defined epitopeor specific binding site 2.Green fluorescent protein : fusion with GFP. 3.His-tag: usually 6 consecutive histidines, which allows purification of the fusion protein by binding to Ni 2+ column. Commonly used in E. coli.

18 Amp r ori pUC18 (3 kb) MCS Lac promoter lacZ’ …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… EcoRISacIKpn I SmaI XmaI BamHI XbaI SalI HincII AccI PstISphI Lac Z’ 1.The ORF of the inserted gene has to be in the same direction and same frame as the lacZ’ 2.A fusion protein between the N-terminal sequence of lacZ and the inserted ORF produced

19 How to make a fusion protein (in pUC18)? ATGATTACGAATTCGAGCTCGGTACCCGGGGATCCTCTAGAGTCGACCTGCAGGCATGCAAGCTT M I T N S S S V P G D P L E S T C R H A S L ATGATTACGAATTCGAGCTCGGTACCCGGGGATCCgatgcggagc..gtgaacggatagCTGCAG M I T N S S S V P G D P M R S..V N G * EcoRI SacI KpnI SmaI XmaI BamHI XbaI SalI HincII AccI PstI SphI Hind Ⅲ Add one nucleotide (g) between BamHI site (GGATCC) and the first codon (ATG) to fuse the two ORFs BamHI: GGATCC PstI: CTGCAG CCTAGG GACGTC From original ORF Inserted ORF The following fusion is wrong: ATGATTACGAATTCGAGCTCGGTACCCGGGGATCCatgcggagc..gtgaacggatagCTGCAG M I T N S S S V P G D P C G G.. *

20 H2 Bacteriophage vectors — Bacteriophage λ 1.Viruses that can infect bacteria kb in length 3.Lytic phase : Replicate and release 4.Lysogenic phase : integrate into host genome

21 5‘-CGGGGCGGCGACCTCG-3’ 3’-GCCCCGCCGCTGGAGC-5’ Cleavage Ligation (during packaging) (after infection) GGGCGGGCGACCTCG-3’ 5’-CG + GC-5’ 3’-GCCCCGCCGCTGGA 5.The phage cos ends ( Linear or circular genome ) Circular form Linear form

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23 H2 Bacteriophage vectors — λReplacement vectors e.g. EMBL3, DASH Replace the nonessential region of the phage genome with exogenous DNA (~20kb)

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25 H2 Bacteriophage vectors — Packaging and infection Replication of phage λin vivo produces long linear molecules with multiple copies of the λ genome. These concatemers are then cleaved at the cos sites, to yield individual λ genomes, which are then packaged into the phage particles. Ligated λ ends which do not contain an insert, or have one which is smaller or larger than the 20kb optimum, are too small or to large to be packaged, and recombinants with two left or right arms are likewise not viable. High infection efficiency (109 recombinants/ug vector DNA, 100-time higher than plasmid)

26 H2 Bacteriophage vectors — Formation of plaques 1.The clear areas within the lawn where lysis and re- infection have prevented the cells from growing. 2.Recombinant l DNA may be purified from phage particles from plaques or from liquid culture.

27 H2 Bacteriophage vectors — λLysogens Genes 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 The E. coli strain BL21(DE3) include the gene for T7 RNA polymerase under control of the lac promoter as a lysogen. The gene can be induced by IPTG, and the polymerase will then transcribe the gene in the expression vector.

29 H2 Bacteriophage vectors — M13 phage vectors E. coli vector; 6.7 kb circular single strand of DNA; 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.

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31 H2 Bacteriophage vectors — Cloning in M13 When the single-stranded form of a fragment is required fragments are subcloned into M13 RF using standard plasmid methods. Cloning  Transfection  Growth  Plaques formation RF recombinant plating on slow like plasmid DNA a cell lawn growth

32 H2 Bacteriophage vectors — Hybrid plasmid-M13 vectors Small plasmid vectors being developed to incorporate M13 functionality. Contain both the plasmid and M13 origins 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.

33 H3 Cosmids, YACs and BACs — Cloning large DNA fragments 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 The limitation of conventional gel electrophoresis: large DNA fragments do not separate, but instead, comigrate, because nucleic acids alternate between globular and linear forms. If the field is applied discontinuously and the direction is also made to vary,the DNA molecules reorient their long axes and takes longer for larger molecules.

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36 H3 Cosmids, YACs and BACs — Cosmid vectors Utilizing the properties of the phage l cos sites in a plasmid vector. The insert can be kb

37 The simplest cosmid vector A normal small plasmid, containing a plasmid origin of replication, a selectable marker, a cos site, a suitable restriction site for cloning.

38 Cloning in a cosmid vector C2XB

39 H3 Cosmids, YACs and BACs — YAC vectors Essential components of YAC vectors : Centromere Telomere Autonomous replicating sequence Amp r for selective amplification and markers

40 YACs c an accommodate genomic DNA fragments of more than 1 Mb, and can be used to clone the entire human genes, but not good in mapping and analysis. YACs have been invaluable in mapping the large-scale structure of large genomes, for example in the Human Genome Project.

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43 H3 Cosmids, YACs and BACs — Selection in S. cerevisiae (1)Growth of yeast on selective media lacking specific nutrients can serve for selection. Auxotrophic yeast mutants are made as host strains for plasmids containing the genes complementary to the growth defect. (2)Saccharomyces cerevisiae selectable markers do not confer resistance to toxic substances

44 H3 Cosmids, YACs and BACs — BAC vectors BAC: Bacterial Artificial Chromosome, 300kb Vectors for large DNA fragments BAC : 300kb PAC: Bacteriophage P1 cloning system , 100kb Cosmid : kb YAC: > 1Mb (1987)

45 H4 Eukaryotic vectors — Cloning in eukaryotes Many applications of genetic engineering require vectors for the expression of genes in diverse eukaryotic species.

46 H4 Eukaryotic vectors — Transfection of eukaryotic cells The take-up of DNA into eukaryotic cells 1.More problematic than bacterial transformation. The plant cell wall must normally be digested to yield fragile protoplasts, which may then take up DNA fairly readily. 2.Much lower efficiency

47 Transfection methods: Calcium phosphate Electroporation Gene gun Microinjection

48 H4 Eukaryotic vectors — Shuttle vectors Most of the eukaryotic vectors are constructed as 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 vectors can be completed in E. coli., before transfer to the appropriate eukaryotic cells.

49 MCS A Shuttle vector

50 H4 Eukaryotic vectors — Yeast episomal plasmids Vectors for the cloning and expression of genes in Saccharomyces cerevisiae. 1.Based on 2 micron (2m) plasmid which is 6 kb in length. One origin Two genes involved in replication A site-specific recombination protein FLP, homologous to Int. Normally replicate as plasmids, and may integrate into the yeast genome. 2. Normally replicate as plasmids, and may integrate into the yeast genome.

51 YEp vector

52 H4 Eukaryotic vectors — Agrobacterium tumefaciens T i plasmid 1. Place the target gene in the T-DNA region of a Ti plasmid, then transform the recombinant Ti plasmid. (Not good because of the crown gall formation) Deletion of the genes in T-DNA that are responsible for crown gall formation. The deleted T-DNA is called disarmed T-DNA shuttle vector. 2. The T-DNA and the remainder of the Ti plasmid are on separate molecules within the same bacterial cell, integration will still take place. Plasmid with recombinant T-DNA can be transformed into the A. tumefaciens cell carrying a modified Ti plasmid without T-DNA.

53 Agrobacterium mediated gene transfer crown gall or tumor

54 Agrobacterium mediated gene transfer

55 H4 Eukaryotic vectors — Baculovirus 1.Infects insect cells 2.The 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. 3.Insect expression system is an important eukaryotic expression system.

56 H4 Eukaryotic vectors — Mammalian viral vectors 1. SV kb, suffers from packaging constraints similar to phage l. 2. Retrovirus Single-stranded RNA genome, which copy to dsDNA after infection. Integrated into the host genome by a transposition-like mechanism. Have some strong promoters for gene expression. Considered as vectors for gene therapy

57 H4 Eukaryotic vectors — Direct gene transfer Genes may be transiently or permanently introduced into cultured eukaryotic cells without the use of vector.

58 Multiple choice questions 1. Blue-white selection is used. A to test for the presence of a plasmid in bacteria. B to reveal the identity of a cloned DNA fragment. C to express the product of a cloned gene. D to test for the presence of a cloned insert in a plasmid. 2. A multiple cloning site. A contains many copies of a cloned gene. B allows flexibility in the choice of restriction enzymes for cloning. C allows flexibility in the choice of organism for cloning. D contains many copies of the same restriction enzyme site.

59 3. Infection of E. coli by bacteriophage λis normally detected by. A resistance of the bacteria to an antibiotic. B the growth of single bacterial colonies on an agar plate. C the appearance of areas of lysed bacteria on an agar plate. D restriction digest of the bacterial DNA. 4. Which vector would be most appropriate for cloning a 150 kb fragment of DNA? A a plasmid. B a λvector. C a BAC. D a YAC.

60 5. Which vector would you chose to express a foreign gene in a plant? A a baculovirus vector. B a retroviral vector. C a Yep vector. D a T-DNA vector.

61 THANK YOU !


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