Microbiology. Zhenmei Lu ( 吕镇梅 ) 2010 Spring-Summer 2017 Spring-Summer

Transcription

1 Microbiology Zhenmei Lu ( 吕镇梅 ) lzhenmei@zju.edu.cn 2010 Spring-Summer 2017 Spring-Summer

2 Chapter 11 Genetic Engineering

3 The fathers of genetic engineering Boyer and Cohen

4 Human insulin The first licensed drug generated using recombinant DNA technology was human insulin, developed by Genentech and Licensed by Eli Lilly and Company.

5 Target genes Cloning vector Enzymes Host

6 Outline Methods for manipulating DNA Gene cloning

7 I. Methods for Manipulating DNA Tools and techniques 11.3 Essentials of Molecular Cloning 11.4 Molecular Methods for Mutagenesis 11.5 Gene Fusions and Reporter Genes

8 Tools and Techniques Restriction Enzymes: Commonly used type II restriction endonucleases cut the DNA within their recognition sequences (palindrome). Most restriction enzymes are homodimeric proteins. Modification enzymes: Methylation. CH 3 CH 3 Single-stranded sticky ends

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10 Tools and Techniques Electrophoresis (Agarose): separation of DNA molecules due to their negatively charged phosphate group. Visualized under UV because of ethidium bromide (EB)

11 Restriction analysis of DNA Molecular cloning Classification of microorganisms Optical mapping of restriction fragments

12 Tools and Techniques Nucleic Acid Hybridization: DNA X DNA (in the gel) southern blot, detect existence of DNA; DNA X RNA (in the gel) northern blot, detect existence of mrna. Highthroughput southern blot: microarray

13 Frederick Sanger Twice a Nobel Laureate in Chemistry

14 Technology changes quickly, but for many years we ve on Sanger s cool trick.

15 12. 2 Sequencing DNA The Sanger Dideoxy Method

16 12. 2 Sequencing DNA Sanger sequencing (Dideoxy)

17 The Sanger Dideoxy Method DNA strand to be sequenced Radioactive DNA primer Add DNA polymerase, mixture of all four deoxyribonucleotide triphosphates; separate into four reaction tubes A small amount of only one dideoxynucleotide triphosphate (ddgtp, ddatp, ddttp, or ddctp) added to each tube and reaction allowed to proceed Reaction products ddgtp ddatp ddttp ddctp G A T C Four different reactions must be run, one with each dideoxynucleotide.

18 The Sanger Dideoxy Method Reaction products separated by electrophoresis on gel and identified by autoradiography 2 1 Sequence reads from bottom of gel as A G C T A A G. Sequence of unknown is 3 T C G A T T C 5 A G C T A A G A portion of a gel containing the reaction products DNA sequences using an automated sequencer and fluorescent labels

19 454 Pyrosequencing 1 DNA samples is broken into single stranded segments; 2 Each fragment is immobilized to a microscopic bead; 3 DNA is amplified by PCR; 4 The beads are put into a fiber-optic plate; 5 The four nucleotides are flowed sequentially over the plate in a fixed order; 6 Sequencing reactions occur.

20 Synthesizing DNA 1Primers (DNA sequencing, PCR); 2Probes (Southern or Northern blots); 3Site-directed mutagenesis

21 Copies of target gene DNA polymerase Target gene(s) Heat Repeat cycle PCR cycle Primers Primer extension Copies of target gene(s) PCR (Polymerase Chain Reaction) Denaturation, annealing, extension Repeat cycle Number of PCR cycles

22 PCR DNA polymerase Escherichia coli Pol III Thermus aquaticus, Taq polymerase, stable at 95 Pyrococcus furiosus, Pfu polymerase, proofreading activity Applications and sensitivity of PCR Cloning genes or sequencing Comparative or phylogenetic analysis Amplify very small amount of DNA Common tool for diagnostic microbiology DNA fingerprinting

23 Applications: Microbial gene expression; Cloning eukaryotic genes Reverse transcriptase PCR

24 Real-time qpcr Amplification can be monitored continuously. Accurately determine the amount of target DNA present in the original sample. Access the abundance of an organism in a sample by quantifying a gene characteristic of that particular organism.

25 Microbial Sidebar Allele 1 Allele 2 EcoRI EcoRI VNTR EcoRI EcoRI Digest DNA, run gel, and blot with probe Gel Two different alleles of a region of a single chromosome VNTR Allele 1 Allele 2 Primer Primer Run PCR reaction, and resolve products on gel The same alleles as (a), but using specific primers to amplify the VNTR segments by PCR

26 11.3 Essential of Molecular Cloning Conventional Cloning

27 11.3 Essential of Molecular Cloning att: Flanking recombination sequence Gateway Cloning

28 DNA library

29 11.3 Essential of Transformant colonies growing on agar surfaces Molecular Cloning Radiolabeled antibodies are used as detection reagents for a protein of interest. Partially lyse cells; add specific antibody; add agent to detect bound antibody in radiolabeled form Replica plate onto membrane filter Lyse bacteria and denature DNA; add RNA or DNA probe (radioactive); wash out unbound radioactivity Nucleic acid probe labeled with radioactive phosphate or a fluorescent chemical group is used to detect nucleic acids containing specific sequences. Autoradiograph to detect radioactivity X-ray film Positive colonies

30 11.4 Molecular Methods for Mutagenesis Site-directed mutagenesis PCR-based

31 1/4 4/8 11/16 26/32

32 PCR-based mutagenesis

33 11.4 Molecular Methods for Mutagenesis Gene X EcoRI cut sites Kanamycin cassette Linearized plasmid Chromosome BamHI cut site Gene X knockout Cut with EcoRI and ligate Cut with BamHI and transform into cell with wild-type gene X Sites of recombination Recombination and selection for kanamycin-resistant cells Gene disruption by double homologous recombination Not very good because it introduces an antibiotic marker Gene Disruption by Cassette mutagenesis

34 11.5 Gene Fusions and Reporter Genes Target gene Promoter Coding sequence Reporter gene Promoter Gene fusion Promoter Coding sequence Cut and ligate Reporter is expressed under control of target promoter Reporter enzyme Substrate Colored product Reporter systems: to assess the target promoter activity by transcriptional fusion; to determine locus of target protein by translational fusion.

35 II. Gene Cloning 11.6 Plasmids as Cloning Vectors 11.7 Hosts for Cloning Vectors 11.8 Shuttle Vectors and Expression Vectors 11.9 Bacteriophage Lambda as a Cloning Vector Vectors for Genomic Cloning and Sequencing

36 11.6 Plasmid as cloning vector (1) Small size; (2) Independent origin of replication; (3) Multiple copy number; (4) Selectable marker. Ampicillin resistance puc base pairs lacz Polylinker Order of restriction enzyme cut sites in polylinker Apo I - EcoR I Ban II - Sac I Acc65 I - Kpn I Ava I - BsoB I - Sma I - Xma I BamH I Xba I Acc I - Hinc II - Sal I BspM I - BfuA I Sbf I Pst I Sph I Hind III lacl Tight cellular control Relaxed cellular control Origin of DNA replication Cloning Vector

37 11.6 Plasmid as cloning vector lacz Insertional inactivation Cells containing the vector with an insert of cloned DNA are white, and cells containing the vector without cloned DNA are blue. AmpR Vector Opened vector Digestion with restriction enzyme Recyclized vector without insert Transform into Escherichia coli and select on ampicillin plates containing Xgal Transformants blue ( -galactosidase active) Foreign DNA Join with DNA ligase Vector plus insert Transformants white ( -galactosidase inactive)

38 11.7 Hosts for Cloning Vectors Prokaryotes Eukaryote Escherichia coli Bacillus subtilis Saccharomyces cerevisiae Well-developed genetics Many strains available Best-known prokaryote Potentially pathogenic Periplasm traps proteins Easily transformed Nonpathogenic Naturally secretes proteins Endospore formation simplifies culture Genetically unstable Genetics less developed than in E. coli Well-developed genetics Nonpathogenic Can process mrna and proteins Easy to grow Plasmids unstable Will not replicate most prokaryotic plasmids Advantages Disadvantages

39 11.7 Hosts for Cloning Vectors Transfection of Eukaryotic cells Transfection, the introduction of DNA into mammalian cells. Phagocytosis ( 吞噬作用 ), animal cells do not have cell walls. Microinjection, using micropipets. Electroporation, exposes host cells to pulsed electrical fields in the presence of cloned DNA. High-velocity microprojectile gun.

40 DNA gun for transfection of eukaryotic cells

41 11.8 Shuttle Vectors Vectors that can replicate and are stably maintained in two (or more) unrelated host organisms. oric Ampicillin resistance t/pa ESM: Eukaryotic selective marker Shuttle vector ESM p CEN: Yeast centromere sequence oriy CEN p t/pa Polylinker (cloning site) t/pa: Transcription termination/polyadenylation

42 11.8 Expression Vectors lacl pse420 Origin of DNA replication trc promoter laco S/D Polylinker (cloning site) T1 T2 Ampicillin resistance High levels of transcription require strong promoters (lac, trp, tac, trc, etc); The transcription is regulated by a repressor protein; Strong transcription terminators are used to halt transcription immediately.

43 11.8 Expression Vectors

44 11.9 Virus Vectors COS Capsid genes J att int xis N QSR COS Replaceable region Wild-type lambda -Gal gene Another substitution Charon 4A (replacement vector) -Gal gene Another substitution Charon 16 (insertional vector) Cloning Vectors (Efficient infection, Cloning capacity)

45 11.9 Virus Vectors Replaceable region R Foreign DNA L Hybrid DNA R L Packaging cloned DNA into phage head cos Digestion with restriction enzymes L R R R Foreign DNA L Ligation with foreign DNA L Hybrid DNA Phage assembly Infective lambda virion Cloning Vectors Cosmid Fosmid

46 11.9 Virus Vectors Cosmids are constructed from plasmids by ligating the cos region to the plasmid DNA. 50 kbp can be inserted; Storage of DNA; No need to transform E. coli; More stable.

47 11.10 Vectors for Sequencing EcoRI SmaI BamHI KpnI XbaI SalI PstI HindIll Polylinker lacp lacz M13 genomic DNA M13: Single strand direct sequence Phage in clear plaques have cloned DNA Phage in blue plaques do not have cloned DNA

48 11.10 Vectors for Sequencing Cloning region Keep copy number low sopb cat Selectable marker sopa 6.7Kb oris repe Required for replication BAC: Bacterial Artificial Chromosome, derived from F plasmid. 300 kbp can be inserted.

49 11.10 Vectors for Sequencing Eukarytotic artificial chromosomes Selectable marker TEL ARS CEN Not I Not I URA3 TEL O INSERTED DNA YAC (1) An origin of DNA replication; (2) Telomeres for replicating DNA at the ends of the chromosome; (3) A centromere for segregation during mitosis. A yeast artificial chromosome containing foreign DNA

50 Thanks for your attention!