PCRExtractionplasmidpET22bpET22b-AnsBAmplifyAnsBgene食Agrosegelelectrophoresis(AGE)testUsingHindIII and NcoI restriction endonucleasedigestplasmid and PCRproductsPurifybothPCRandplasmidpET22bdigestedproductsCultivateE.coliBL21by PCR extraction Kit 几PurifiedproductslinkedbyT4PreparationofcompetentcellbyDNAligase(16°Covernight)using CaCI2methodE.coliBI21transfomationChapter I几ScreentherecombinantbacteriaonLB solid medium(Amp100)1Inoculate single colony in the LB liquid mediumExtractiontheplasmidandconfirmwithAGE
Chapter I pET22b-AnsB PCR Amplify AnsB gene Extraction plasmid pET22b Agrose gel electrophoresis (AGE) test Using Hind III and Nco I restriction endonuclease digest plasmid and PCR products Purify both PCR and plasmid pET22b digested products by PCR extraction Kit Purified products linked by T4- DNA ligase(16℃ overnight) Cultivate E. coli BL21 Preparation of competent cell by using CaCl2 method E. coli Bl21 transfomation Screen the recombinant bacteria on LB solid medium (Amp100) Inoculate single colony in the LB liquid medium Extraction the plasmid and confirm with AGE
InoculaterecombinantbacteriawithpET22b-AnsBintheLBliquidmediumUsingIPTGtofinishinducibleexpressionCentrifugation and collectionofrecombinantbacteria(6000g,4°C,15min)Solidacetatebuffer suspended and usingosmotic shocktoreleasetargetproteinAmmonium sulfatefractionation:90% saturation, collect designed proteinDialysisChapter IISeparatetargetproteinwithNicolumnTest enzyme activity and contentof protein: SDS-PAGE electrophoretic analysisAnalysis drawing enzyme activity curve,protein seperation curve
Inoculate recombinant bacteria with pET 22b-AnsB in the LB liquid medium Using IPTG to finish inducible expression Centrifugation and collection of recombinant bacteria (6000g, 4℃, 15min) Solid acetate buffer suspended and using osmotic shock to release target protein Ammonium sulfate fractionation: 90% saturation, collect designed protein Dialysis Separate target protein with Ni column Test enzyme activity and content of protein: SDS-PAGE electrophoretic analysis Analysis drawing enzyme activity curve, protein seperation curve Chapter II
Ava l(158)Xho l(158)Not l(166)Eagl(166)Hind ill173),Bpu1102 I(80)Sal l(179)Saci(190)EcoRI(192)BamH(198)NcO(220)Msc I(225)BseR(280)Dra ll(5251)BspM I(208)Nde l(288)Xba l(328)Bgl I(302) oigin(5027-5482)SgrA l(433)Sph i(580)PfIM (696)ApaB l(7ee)Sca l(4588)Ap(4038-489Pvu l(4478)Mu I(1114)Bcll(1128)Pst (4353)lacl（764-1843)BstEIl(1295)Bmgl(1323)Bsal(4160)Apal(1325)m1105 (4108)pET-22b(+)(5493bp)BssHIl(1525)Plasmid profile ofHpa I(1620)pET22bAwNl(3631)OPshA l(1059)Psp5Il(2221)BspLU11(3215)Bpu10l(2321)Sapl(300g)Bst1107((29e6)BspGI(2741)Tth111 (29e0)
Plasmid profile of pET22b
UpET-22b(+)cloning/expression regionT7promoterprimer#00348-317promoterBgilllacoperatorXbalrbsAGATCTCCATCCCGCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGABspMIMsclNcolpelB leaderNdelBamHIEcoRISacTATACATATCAAATACCTGCTGCCGACCCCTGCTGCTCGTCTCCTGCTCCTCGCTGCCCACCCGCCGATGCCCATCGATATCGGAATTAATTCGGATCCGAATTCGAGCTCCMetLysTyrLeuLeuProThrAlaAlaAlaGlyLeuLeuLeuLeuAlaAlaGlnProAlaMetAlqMetAsplleGlyl leAsnSerAspProAsnSerSerSerEag!signalpeptidaseAvarHis-TagBpu/11021SallNotlXhoiHind IllGTCGACAAGCTTGCGCCCGCACTCGAGCACCACCACCACCAC CACTGAGATCCGGC.TGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGC TGCCACCGCTGAGCAATAACValAspLysLeuAlaAlaAlaLeuGluHisHisHisHisHisHisEnd17terminatorprimer#ee337-3T7terminatorTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG
pET-22b(+)cloning/expression region
#Chapter 1The Construction of Recombinant AsparaginaseII Engineering Bacteria
Chapter 1 The Construction of Recombinant Asparaginase II Engineering Bacteria