转化简短发展史和基础实验流程

1、Literatures ReportA Brief History of Transformation and Basic ProtocolsOctober 17, 2016Speaker： %Mentor： %Contents1 Introduction2 Brief History 3 Basic Protocols 4 DiscussionImages copyright belong to Wikipedia This process of bacterial cell 2(the square one) taking up the new genetic material which

2、 is belong to bacterial cell 1(the elliptical one)is called Transformation.1928-1944 Frederick Griffith-Transforming principle Oswald Theodore Avery Transformation(1944)1970-1983 Morton Mandel and Akiko Higa-Bacteriophage Stanley Norman Cohen-Plasmid DNA (1972) Douglas Hanrahan-Method of transformat

3、ion1980s-1990sCaCl2（E.coli）21thPotter, Huntington; Heller, Richard-Transfection by Electroporation(2003)Calum Johnston-Bacterial transformation(2014)E.Neumann -Eelectrical field（1982）E.Neumann -Induced membrane pores electroporationJames C Weaver,etal-Theory of electroporationElectroporationPrincipl

4、esFred Griffith. The significance of pneumococcal typesJ. Hyg, 1928, 27: 113159. Lederberg Joshua.TheTransformationofGeneticsbyDNA:AnAnniversaryCelebrationofAVERY,MACLEODandMCCARTY(1944)inAnecdotal,HistoricalandCriticalCommentariesonGenetics,1994,TheRockefellerUniversity,NewYork,NewYork10021-6399.格里

5、菲斯肺炎双球菌转化实验：“转化因子”奥斯瓦尔德埃弗里肺炎链球菌转化；DNA为遗传物质大肠杆菌在CaCl2作用下吸收噬菌体“” Cohen Stanley N,Chang Annie,Hsu Leslie.Nonchromosomal Antibiotic Resistance in Bacteria: Genetic Transformation of Escherichia coli by R-Factor DNAJ.Proceedings of the National Academy of Sciences,1972,69 (8): 21104.大肠杆菌在CaCl2作用下有效转化质粒Ma

6、ndel Morton,Higa Akiko.Calcium-dependent bacteriophage DNA infectionJ. Journal of Molecular Biology,1970,53 (1): 159162. Douglas Hanahan. Studies on transformation of Escherichia coli with plasmidsJ.Journal of Molecular Biology,1983,166 (4): 557580.1、目标菌株置于0冰浴进行孵育；2、二价金属阳离子（Mg2+、Ca2+）对转化有促进作用；3、二甲基亚

7、砜、二硫苏糖醇及乌洛托品钴的作用；4、转化效率随质粒的增大呈线性递减。CaCl2作用下的质粒转化规律及条件的优化IF：4.517（15-16）E Neumann,M Schaefer-Ridder,Y.Wang,P.H.Hofschneider .Gene transfer into mouse lyoma cells by electroporation in high electric fieldsJ.The EMBO Journal, 1982,1 (7): 8415.电穿孔法增加细胞膜（鼠）的通透性，可使大分子物质进入细胞；但首批实验产生的稳定转化子并不多。EMBO：European

8、Molecular Biology Organization (IF:9.643) （15-16）Sugar, Istvan P.; Neumann, Eberhard.Stochastic model for electric field-induced membrane pores electroporationJ.Biophysical Chemistry,1984,19 (3): 21125.电场下细胞膜电穿孔法的随机模型IF：2.363（15-16）Bioelectrochemistry & Bioenergetics, 1996, 41(2):135-1601、高压电场下，

9、平面脂双分子膜结构不稳定的关键机械特征在于电穿孔造成的不可逆影响；2、某些特定平面模结构及细胞膜，有剧烈的、可逆性的电场变化特征；3、运输分子（蛋白）的相关特性起特异作用。IF：3.556（15-16）2022-3-169Potter Huntington,Heller Richard. Transfection by Electroporation. Current Protocols in Molecular BiologyM. 2003. CHAPTER: Unit9.3.电转化实验流程，刊载于03年出版的现行分子生物技术通用指南，流程共13页，详细介绍了电转化实验的各个过程，适用于绝大

10、多数细胞（侧重于哺乳动物细胞及植物细胞）。基础实验流程包括：1 Materials2 Prepare the cells for electroporation3 Add DNA and electroporate the cells4 Culture and harvest the transfected cells Johnston C, Martin B, Fichant G, Polard P, Claverys JP. Bacterial transformation: distribution, shared mechanisms and divergent controlJ. N

11、at. Rev. Microbiol,2014,12 (3): 18196.Nature Reviews Microbiology IF：24.727（15-16）1、至2014年，经报道确认，在自然条件下产生感受态并可发生转化的细菌有近80种（待查是否包含嗜铜菌）；2，可根据现有可转化类别，推测未报道或新菌种是否能被转化。2022-3-1611Basic protocol of Transformation Note: Addgene owns the pictureSpeciesStepsE. coli JM105【1】E. Coli【2】 Strains preparationBacte

12、ria were grown in liquid media at 37 C to mid-log phase (optical density of 0.3 at 600 nm) with shaking. E. coli wre Grown in LB liquid media at 37 C for 10-12h.; Inoculated it to new media in one percent. Competent cell preparationWashed once in transformation buffer (300 mM sucrose, 7 mM sodium ph

13、osphate, pH 7.4, 1 mM MgC12) and resuspended intransformation buffer to yield 109 colony forming units (cfu)/ml.Washed twice in transformation buffer (10% glycerin) and resuspended in10% glycerin with a volume about 0.75% of the initial volume Mixed cells with plasmid in a certain proportionThe cell

14、 suspension (1 ml) was mixed with 200 ngof pKT231-DNA, incubated for 30 min on ice, an aliquotwas removed for controls and 800 L of the mixture were subjected to electroporation (one pulseat 6250 V/cm using the 25 gF capacitor) in a cooled cuvette. The cell suspension (150 l) was mixed with about 1g

15、of P1000-DNA, incubated for 15 min on ice;One pulse at 200 , 25 Fd, 2.5 kV with a cooled 2 mm cuvette.Time last to 3 seconds. Incubated and Choosed positive cloneTe mixture was kept for 20 min on ice, diluted in theoriginal growth medium, incubated 37C for 45 min and various amounts plated on origin

16、al medium plates containing 50 g kanamycin/ml. Incubated 37C for 1h and various amounts plated on original medium plates containing appropriate concentration of antibiotics1 Reinhard Wirth,Anita Friesenegger,et al.Transformation of various species of gram-negative bacteria belonging to 11 different genera by electroporationJ.Mol Gen Genet.1989,216(1):175-177NOTE：“【2】”为生科院XX老师的Manuscript protocol.2022-3-1612ProtogeneticPlasmidE.coli WA803 pTR102:luxAB ,tetr,kmrSuspension desal