球形芽孢杆菌脂肪酶基因的克隆表达.pptVIP

由于相同菌种脂肪酶基因两端保守性不太高，根据芽孢杆菌脂肪酶蛋白序列保守区设计兼并引物，如下：扩增产物长度496bp. sense bao 5TC(G/T)AG(C/T)AACTGGGA therm.Tm 54（参照引物单） (C/T)CG(G/T)GC(G/A)TG 3 antisense bao 5CCCATTG(G/A)TC(G/C) therm.Tm 45（参照引物单） A(G/A)(C/T)TTAAAATC 3 进行梯度PCR实验，退火温度从45到54℃，递减1℃，共10个反应管，并分别设上下游单引物对照，及空白对照。 上游引物 下游引物 引物对 空白 方案1：参照文献中的引物，（酶切位点） 方案2：菌种理化性质鉴定 方案3：用已分离的其他菌种做材料，重新扩增lipase 基因 * * Lipase gene analysis method Restriction analysis PCR analysis A Bacillus sphaericus strain (205y) that produces an organic solvent-tolerant lipase. The gene for the lipase was recovered from a genomic library. Phylogenetic analysis Expression in Escherichia coli under the control of the lacZ promoter Organic solvent-tolerance Chomosomal DNA was extracted, and partially digested with Sau3AI. The reaction was optimized so that most of the fragments were between 2 and 10 kb long. Sau3AI : 5’…ˇGATC…3’ 3’…CTAGˇ…5’ PUC19 BamH I: 5 ..G ˇ GATCC.. 3‘ 3’..CCTAGˇG..5’ Lipase-producing colonies induce① the formation of haloes on tributyrin agar and② blue zones on triolein agar. ③Recombinant clones were also screened on Rhodamine B agar. A clone was found to contain a 9 kb DNA fragment after BamHI digestion and gel electrophoresis of the isolated plasmid (Fig. 1). Restriction mapping of the putative lipase gene(9 kb DNA fragment partially digested with Sau3AI,205y pLIP ) using EcoRI, XbaI, HindIII, SacI, KpnI,SmaI, BamHI, and BglI SacI : two bands (900 bp and 4400 bp) HindIII :three bands (650 bp, 550 bp and 4200 bp). ~2.9kb sacI/Hind III 测序 Recombinant clones carrying the 900-bp, 800-bp, 650-bp, and 550-bp fragments did not produce a halo on tributyrin-amp plate.A recombinant carried the 2 kb SacI/HindIII fragment gave a positive result on tributyrin-amp plates and produced a blue zone on triolein-amp Victoria Blue agar plate. This plasmid was n