Microbiology 微生物学 Spring-Summer

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2 Microbiology 微生物学 2017 Spring-Summer

3 Relevant Information and Resources Course slides can be found at 教学工作 Course-related questions will be answered through s. Textbook: Brock Biology of Microorganisms (13 th )

4 Chapter 11 Genetic Engineering I DNA Manipulation

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

6 Restriction enzymes

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

8 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

9 Tools and Techniques Foreign DNA Cut with restriction enzyme Sticky ends Add vector cut with same restriction enzyme Vector Cloned DNA Introduction of recombinant vector into a host Conventional Cloning Add DNA ligase to form recombinant molecules 1. Limitations in sites 2. Preferences in cloning small Fragments 3. The larger the plasmid, the harder the cloning

10 Tools and Techniques Gateway Cloning

11 Tools and Techniques lacz 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 AmpR Vector Opened vector Digestion with restriction enzyme Foreign DNA Join with DNA ligase lacl Origin of DNA replication Cloning Vector Recyclized vector without insert Transform into Escherichia coli and select on ampicillin plates containing Xgal Transformants blue ( -galactosidase active) Vector plus insert Transformants white ( -galactosidase inactive)

12 Cloning Finding the right clone 1. Looking for the right sequence - hybridization 2. Looking for the right protein hybridization 3. Looking for the right phenotype based on phenotype indicator

13 Cloning Finding the right clone Transformant colonies growing on agar surface Replica-plate onto membrane filter Partially lyse cells; add specific antibody; add agent to detect bound antibody in radiolabeled form Lyse bacteria and denature DNA; add RNA or DNA probe (radioactive); wash out unbound radioactivity Autoradiograph to detect radioactivity X-ray film Positive colonies

14 Source Clone into single-stranded vector Mutagenesis Single-stranded DNA from M13 phage Site-directed mutagenesis Base-pairing with source gene Add synthetic oligonucleotide with one base mismatch Extend single strand with DNA polymerase Clone and select mutant Transformation and selection

15 Mutagenesis Site-directed mutagenesis

16 Gene X EcoRI cut sites ( ) Kanamycin cassette Mutagenesis Cut with EcoRI and ligate Cassette mutagenesis BamHI cut site Cut with BamHI and transform into cell with wild-type gene X Linearized plasmid Chromosome Sites of recombination Recombination and selection for kanamycin-resistant cells Gene X knockout

17 Gene fusions Reporter genes: LacZ, GFP, and many more

18 Gene fusions Transcriptional/translational fusions Target gene Promoter Coding sequence Reporter gene Promoter Gene fusion Promoter Coding sequence Cut and ligate Reporter is expressed under control of target gene promoter Reporter enzyme Substrate Colored product

19 II Gene cloning

20 Cloning lacz 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 AmpR Vector Opened vector Digestion with restriction enzyme Foreign DNA Join with DNA ligase lacl Origin of DNA replication Cloning Vector Recyclized vector without insert Transform into Escherichia coli and select on ampicillin plates containing Xgal Transformants blue ( -galactosidase active) Vector plus insert Transformants white ( -galactosidase inactive)

21 Host for cloning Bacteria Eukaryote Escherichia coli Bacillus subtilis Saccharomyces cerevisiae Well-developed genetics Many strains available Best known bacterium 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 bacterial plasmids Advantages Disadvantages

22 Host for cloning phgm01 Many hosts require special features.

23 Transfection of eukaryotic cells Before gas release After gas release Plunger Helium gas Gas vent Disc Microprojectiles with transfecting nucleic acid Fine screen Rough screen Target tissue

24 Shuttle Vectors Prokaryotic selectable marker Ampicillin resistance oric for replication in E. coli Transcription terminator/poly-a t/pa ESM Promoter Eukaryotic selectable marker oriy for replication in Yeast Yeast centromere sequence CEN Promoter t/pa Polylinker (cloning site)

25 Expression vector laci pse420 Origin of DNA replication trc promoter laco S/D Polylinker (cloning site) T1 T2 Ampicillin resistance Promoters often used in expression vector: Ntative: Plac, Ptrp, Synthetic: Ptac, Ptrc from Plac and Ptrp Virus: P L from, P T7 from T7 phage Multiple terminators: prevent reading through

26 Expression vector

27 Expression vector

28 Expression System Induce lac promoter with IPTG T7 RNA polymerase Gene product lac promoter lac operator Gene for T7 RNA polymerase T7 promoter pet plasmid Cloned gene Chromosome lacl Host cells modified to work with pet expression vectors: T7 promoter: under the control of Plac; laci are on both chromosome and plasmid

29 Virus vector as a vector 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)

30 Virus vector R L Replaceable region as a vector cos Digestion with restriction enzymes R R Packaging cloned DNA into phage head Foreign DNA L Ligation with foreign DNA L Hybrid DNA Large DNA fragment No insertion, no packaging High efficiency for DNA transfer by transduction L R Phage assembly Infective lambda virion

31 Cosmid vector Plasmid + Cos It is a plasmid Stable storage Large DNA fragment No insertion, not packaging High efficiency for DNA transfer by transduciton

32 Vectors for genomic work BamHI SmaI EcoRI KpnI XbaI SalI PstI HindIII lacp Polylinker lacz M13 genomic DNA Phage in clear plaques have cloned DNA Phage in blue plaques do not have cloned DNA M13 phage 5-kb Single strand for direct sequencing

33 Vectors for genomic work Polylinker (Cloning site) sopb cat Selectable marker Keep copy number low sopa BAC oris 6.7 kb can hold up to 300 kb Host: defective in recombination repe Required for replication Bacterial Artificial Chromosome: derived from F plasmid

34 Vectors for genomic work Selectable marker TEL ARS CEN NotI NotI URA3 INSERTED DNA YAC TEL Yeast Artificial Chromosome (10 kb): can hold up to 800kb

35 Chapter 12 Microbial Genomics I Genomes and Genomics

36 Prokaryotic Genomes Genome size and ORFs

37 Genome sequencing Cloning region Keep copy number low sopb cat Selectable marker sopa BAC oris repe Required for replication BAC: Bacterial Artificial Chromosome, derived from F plasmid 300kbp can be inserted

38 Frederick Sanger Twice a Nobel Laureate in Chemistry

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

40 Sanger s Trick

41 Sanger s Trick DNA chain Direction of chain growth Base Base No free 3 -OH, replication will stop at this point

42 Sanger s Trick Sanger sequencing (Dideoxy)

43 Sanger s Trick 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 Sanger sequencing (Dideoxy) G A T C 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

44 New Generation Sequencing Technology

45 454 Pyrosequencing

46 DNA Sequencing

47 DNA Sequencing

48 DNA Sequencing

49 DNA Sequencing

50 PyroSequencing

51 DNA Sequencing

52 DNA Sequencing

53 DNA Sequencing

54 DNA Sequencing

55 DNA Sequencing