e微生物学南京师范大学英文

1、MICROBIOLOGYChapter 8. Microbial GeneticsDefinition 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 expressed dur

2、ing 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 DNA TECH

3、NIQUESPRODUCTIONS 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” genetic

4、 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

5、production 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. Thi

6、s is very important 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 RECOMBIN

7、ANT DNA TECHNIQUESPRODUCTIONS 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

8、 Talaro, in FOUNDATIONS IN MICROBIOLOGY, 2nd ed, 2005 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

9、recombinant DNA molecules.In 1973, plasmid 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 MICROBIOL

10、OGY, 5th edRestriction enzyme digestion of DNA5-NNNNNNNNGAAT 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

11、 SciI2. DNA Ligases e.g. T4 DNA ligase5-NNNG AAT T CNNN -33-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-

12、5-5-3. Cloning vectorsCloning vector is a DNA molecule that can replicate, 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 g

13、eneOriAmpRForeign geneMCSVectors carry genes into host cells4. Reverse transcriptase 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-depe

14、ndent DNA polymerase that uses a viral RNA genome as a template to form a DNA 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-AAAAAA

15、AAAA-TTTT TTT-53 3 3TTTT TTT-5cDNAmRNARNaseHRT+dNTPSynthesis of single-stranded cDNAInvented 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

16、h at 95CAllow to perform in machine, thermal cyclerT (C) 90 70 50Time (min)Cycle 1 Cycle 2 Cycle 3 CycleMain topics:BREEDING BY INDUCED MUTATIONBREAKTHROUGHS LEADING TO RECOMBINANT DNA TECHNOLOGYSTRATEGIES OF USING RECOMBINANT DNA TECHNIQUESPRODUCTIONS OF RECOMBINANT PROTEINSGENETIC MODIFICATION OF

17、ORGANISMSDefinitionGenetic engineering: A group of techniques for manipulating DNA outside 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

18、:BREEDING BY INDUCED MUTATIONBREAKTHROUGHS LEADING TO RECOMBINANT DNA TECHNOLOGYSTRATEGIES OF 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. St

19、rong promoter for transcription;iv. Optimized translation; v. Optional regulation;vi. Easy cultivation.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

20、Recombinant of lac & trp, Ptac Promoter from phage T7 PL from E.coli phage l PHsh from E.coliInduction of expression in E. coliv. Optional regulation PL +1 RBS FGTerminator 3040 mRNAHeat shock* ts repressor* A temperature sensitive protein encoded by mutant cI857(ts)Induction of gene expression

21、in PL vector T7 PromoterFGHost cellChromosomeVectorvectorT7 RNApolymeraseGene expression controlled by T7 promoterPlac/PLA new vector controlled by PHsh in E. coliHeat shock. .s32 molecules Recombinant protein.Transformantvi. Easy cultivationMain topics:BREEDING BY INDUCED MUTATIONBREAKTHROUGHS LEAD

22、ING TO RECOMBINANT DNA TECHNOLOGYSTRATEGIES OF USING RECOMBINANT DNA TECHNIQUESPRODUCTIONS OF RECOMBINANT PROTEINSGENETIC MODIFICATION OF ORGANISMSDefinitionMetabolic (pathway) engineering is the use of molecular techniques (or recombinant DNA techniques) to improve the efficiency of pathways that s

23、ynthesize specific products.- L M Prescott et al., in MICROBIOLOGY, 5th edFermentation of ethanol by Zymomonas mobilisEntner-Doudoroff 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

24、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/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 r

25、ecombinant 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. Definition review Genetics is the science of heredity; it i

26、s 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 expressed during the development of an individual.- Stanley R Maloy et al., in MICROBIAL GENETICS, 1994MICROBIOLOGYChapter 8. Microbial GeneticsIV. Applications of Microbial GeneticsInduced mutation ofPenicillium chrysogenumBreeding of strain NRRL-B25 for production of penicillinStrain Mutagen Yield (U/ml)