1、MICROBIOLOGYChapter 8.Microbial Genetics by Weilan ShaoDefinition review Genetics is the science of heredity;it is concerned with the physical and chemical properties of the hereditary material,how this material is transmitted from one generation to the next,and how the information it contains is ex
2、pressed during the development of an individual.-Stanley R Maloy et al.,in MICROBIAL GENETICS,1994MICROBIOLOGYChapter 8.Microbial GeneticsIV.Applications of Microbial GeneticsMain topics:BREEDING BY INDUCED MUTATIONBREAKTHROUGHS LEADING TO RECOMBINANT DNA TECHNOLOGYSTRATEGIES OF USING RECOMBINANT DN
3、A TECHNIQUESPRODUCTIONS OF RECOMBINANT PROTEINSGENETIC MODIFICATION OF ORGANISMSMain topics:BREEDING BY INDUCED MUTATIONBREAKTHROUGHS LEADING TO RECOMBINANT DNA TECHNOLOGYSTRATEGIES OF USING RECOMBINANT DNA TECHNIQUESPRODUCTIONS OF RECOMBINANT PROTEINSGENETIC MODIFICATION OF ORGANISMSA“classical”gen
4、etic techniquefor strain breeding Physical agentsMutagenic agentse.g.X-rays,g-rays,UV Chemical mutagense.g.base analogs,nitrous acid,alkylating,arylating agentsUVL VLPhotolyaseUV Damage&photoreactivation of DNAEXAMPLEInduced mutation ofPenicillium chrysogenumBreeding of strain NRRL-B25 for productio
5、n of penicillinStrain Mutagen Yield(U/ml)TimeNRRL-B25 250 1943NRRL-X1612 X-rays 500 1943NRRL-Q176 UV light 850 1945NRRL-WIS-47-1564 UV light 850 1947-50000 1977Advantages Industrial strain can be improved to a good level of production before related genetic information is obtained.This is very impor
6、tant for fermentation of metabolites,especially for 2nd metabolites.DisadvantagesRelatively minor modifications can be obtained.The nature of mutations is not known.Main topics:BREEDING BY INDUCED MUTATIONBREAKTHROUGHS LEADING TO RECOMBINANT DNA TECHNOLOGYSTRATEGIES OF USING RECOMBINANT DNA TECHNIQU
7、ESPRODUCTIONS OF RECOMBINANT PROTEINSGENETIC MODIFICATION OF ORGANISMSAn overview Biotechnology has been greatly advanced by the introduction of recombinant DNA technologies because organisms can now be greatly modified to accomplish goals that were previously impossible.-Kathleen P Talaro,in FOUNDA
8、TIONS IN MICROBIOLOGY,2nd ed,2019 Breakthroughs in rDNA technology In 1970,Arber,W.&H.Smith reported microbial enzymes cut dsDNA at specific site.In 1970,Temin&Baltimore found reverse transcriptase from retroviruses.In 1972,Jackson et al.successfully generated recombinant DNA molecules.In 1973,plasm
9、id vector was used by Cohen&Boyer for gene cloning.1.Restriction enzymesRestriction enzymes are enzymes produced by host cells that cleaves virus DNA at specific points,and thus protect the cells from virus infection.-L M Prescott et al.,in MICROBIOLOGY,5th edRestriction enzyme digestion of DNA5-NNN
10、NNNNNGAAT TCNNNNNNNNNNN-33-NNNNNNNNCT TAAGNNNNNNNNNNN-5 dsDNAA sequencerecognizedby EcoRICut5-NNNNNNNNG3-NNNNNNNNCT TAAAAT TCNNNNNNNNNNN-3 GNNNNNNNNNNN-5Type II restriction enzymesPalindrome sequences:Cut to produceSticky ends:orBlunt ends:XhoI SciI2.DNA Ligases e.g.T4 DNA ligase5-NNNG AAT T CNNN-33
11、-NNNC TTAA GNNN-55-NNNG AAT T CNNN-33-NNNC TTAA GNNN-5LigasionLigasion of heterogeneous DNANNNNNNNNGNNNNNNNNCTTAAAATTCNNNNNNNNNNN-3TTAAGNNNNNNNNNNN-5NNNNNNNNGAATTCNNNNNNNNNNN-3NNNNNNNNCTTAAGNNNNNNNNNNN-5Annealing LigationP-OH-P-OH3-3-5-5-3.Cloning vectorsCloning vector is a DNA molecule that can rep
12、licate,and is used to transport a piece of inserted foreign DNA into a recipient cell.It may be a plasmid,phage,cosmid or artificial chromosome.-L M Prescott et al.,in MICROBIOLOGY,5th ed.Recombination of plasmid and foreign geneOriAmpRForeign geneMCSVectors carry genes into host cells4.Reverse tran
13、scriptase It is best known for its role in replication of the AIDS virus.It is a valuable tool for converting mRNA into DNA.cDNA cloning simplified the management of eukaryotic genes.Reverse transcriptase(RT)is an RNA-dependent DNA polymerase that uses a viral RNA genome as a template to form a DNA
14、copy;this is a reverse of the normal flow of genetic information,which proceeds from DNA to RNA.-L M Prescott et al.,in MICROBIOLOGY,5th ed5AAAAAAAAAA-AAAAAAAAAA-TTTT TTT-53Poly-A tailOligo-dT primer5AAAAAAAAAA-AAAAAAAAAA-TTTT TTT-53 3 3TTTT TTT-5cDNAmRNARNaseHRT+dNTPSynthesis of single-stranded cDN
15、AInvented by Kary Mullisgroup in the 1980s.To next cycleExtensionAnnealingDenaturation5.Polymerase Chain Reaction(PCR)Heat-stable DNA polymeraseFrom extremophilese.g.Taq polymerase from Thermus aquatcusHalf-life 2 h at 95 CAllow to perform in machine,thermal cyclerT(C)90 70 50Time (min)Cycle 1 Cycle
16、 2 Cycle 3 CycleMain topics:BREEDING BY INDUCED MUTATIONBREAKTHROUGHS LEADING TO RECOMBINANT DNA TECHNOLOGYSTRATEGIES OF USING RECOMBINANT DNA TECHNIQUESPRODUCTIONS OF RECOMBINANT PROTEINSGENETIC MODIFICATION OF ORGANISMSDefinitionGenetic engineering:A group of techniques for manipulating DNA outsid
17、e the organism from which it was obtained,and reintroducing the recombinant or modified DNA into another cell where it will exert its effects.-John L.Ingraham et al.,inINTRODUCTION TO MICROBIOLOGYMain topics:BREEDING BY INDUCED MUTATIONBREAKTHROUGHS LEADING TO RECOMBINANT DNA TECHNOLOGYSTRATEGIES OF
18、 USING RECOMBINANT DNA TECHNIQUESPRODUCTIONS OF RECOMBINANT PROTEINSGENETIC MODIFICATION OF ORGANISMSAdvantages in production of recombinant proteini.Use of modified gene;ii.Increase of gene copies;iii.Strong promoter for transcription;iv.Optimized translation;v.Optional regulation;vi.Easy cultivati
19、on.A easy way to modify a geneYYYXXXYYYYYYXXXXXXYYYAAATTT CCCAAAYYYXXXTTTAAA GGGTTTXXXBlunting Kination LigationPCRi.Use of modified geneii.,iii.&iv.In central dogma lac Promoter from E.coli Recombinant of lac&trp,Ptac Promoter from phage T7 PL from E.coli phage l PHsh from E.coliInduction of expres
20、sion in E.coliv.Optional regulationPL +1 RBS FGTerminator3040 mRNAHeat shock*ts repressor*A temperature sensitive protein encoded by mutant cI857(ts)Induction of gene expression in PL vectorT7 PromoterFGHost cellChromosomeVectorvectorT7 RNApolymeraseGene expression controlled by T7 promoterPlac/PLA
21、new vector controlled by PHsh in E.coliHeat shock.s32 molecules Recombinant protein.Transformantvi.Easy cultivationMain topics:BREEDING BY INDUCED MUTATIONBREAKTHROUGHS LEADING TO RECOMBINANT DNA TECHNOLOGYSTRATEGIES OF USING RECOMBINANT DNA TECHNIQUESPRODUCTIONS OF RECOMBINANT PROTEINSGENETIC MODIF
22、ICATION OF ORGANISMSDefinitionMetabolic(pathway)engineering is the use of molecular techniques(or recombinant DNA techniques)to improve the efficiency of pathways that synthesize specific products.-L M Prescott et al.,in MICROBIOLOGY,5th edFermentation of ethanol by Zymomonas mobilisEntner-Doudoroff
23、 pathwayPyruvateAcetaldehyde+CO2EthanolPyruvatedecarboxylaseAlcoholdehydrogenaseE.coli 的代谢工程 EMP途径乳酸 丙酮酸乙醛 甲酸 乙醇脱氢酶CO2+H2 乙酰辅酶A乙醇 磷酸转移酶 乙醛脱氢酶乙酰磷酸乙醛 乙酸激酶 乙醇脱氢酶乙酸乙醇乳酸脱氢酶丙酮酸脱羧酶乙醇操纵子设计宿主菌株的选择基因的整合表达水平的筛选条件优化等乙醇发酵的代谢工程Production of ethanol from corn fiber hydrolysateStrain Yield Recovery Rate （g/l）（%）（g/
24、l/h）Sacharomyces 1400 21.0 98 1.60Z.mobilis CP4 22.6 88 1.04E.coli KO11 34.7 80 1.16EXAMPLEMetabolic engineered strainsSummeryFirst breakthroughs leading to recombinant DNA technology were discoveries of microbial enzymes which cut,ligate,and synthesize DNA.Genetic engineering has greatly advanced biotechnology,and organisms can be modified to accomplish goals that were previously impossible.
