[Objective] The aim of the research was to study the effect of the ice bath time after heat shock and the incubation time on the transformation efficacy,and to establish a simple and quick transformation method.[Metho...[Objective] The aim of the research was to study the effect of the ice bath time after heat shock and the incubation time on the transformation efficacy,and to establish a simple and quick transformation method.[Method]Competent cells were prepared with two buffer solutions;with the ice bath time after heat shock and the recovery time as the variables,the relationship between these two factors and transformation efficacy was studied.[Result]The transformation efficacy was the best when the ice bath time was 2 min and the recovery time was 30 or 40 min;when the ice bath time and the recovery time was 0 min,a certain amount of transformants still could be obtained.[Conclusion]The ice bath time after heat shock and the recovery time had certain impact on transformation efficacy,but they were not the decisive factors.Therefore,in the general transformation experiment,these two steps could be omitted.展开更多
基金Supported by Foundation for Returned Scholars of Hebei Province(2010)Research Fund for the Doctoral Program of Hebei Normal University(L2009B13)~~
文摘[Objective] The aim of the research was to study the effect of the ice bath time after heat shock and the incubation time on the transformation efficacy,and to establish a simple and quick transformation method.[Method]Competent cells were prepared with two buffer solutions;with the ice bath time after heat shock and the recovery time as the variables,the relationship between these two factors and transformation efficacy was studied.[Result]The transformation efficacy was the best when the ice bath time was 2 min and the recovery time was 30 or 40 min;when the ice bath time and the recovery time was 0 min,a certain amount of transformants still could be obtained.[Conclusion]The ice bath time after heat shock and the recovery time had certain impact on transformation efficacy,but they were not the decisive factors.Therefore,in the general transformation experiment,these two steps could be omitted.
基金Supported by National Natural Science Foundation of China(No.31300601)PUMC Youth Fund(No.33320140186)+1 种基金Natural Science Foundation of Hebei Province(No.C2015205212)Research Funding of Hebei Normal University(No.L2012Z12)~~
文摘丙氨酸脱氢酶可逆催化丙氨酸脱氨生成丙酮酸,在氨基酸和酮酸的合成及代谢中至关重要.本研究通过PCR从巨大芽孢杆菌WSH-002中克隆并构建了丙氨酸脱氢酶基因(aldBM066)的原核表达载体,经原核表达后,采用Ni-NTA亲和层析法和阴离子交换色谱法纯化获得蛋白AldBM066,在289 K下以座滴法进行晶体生长条件筛选和制备.通过对蛋白质结晶条件的筛选,最终在蛋白质浓度为15 mg/mL及含有0.1 mol/L乙酸钠(p H 5.0)和2.4 mol/L甲酸钠的缓冲液中获得了理想的蛋白质晶体,晶体大小约为210μm×180μm×150μm,X-射线衍射数据显示,该蛋白质晶体衍射分辨率为2.88,空间群为三方晶系,晶胞参数为a=b=118.71,c=150.51,α=β=90°,γ=120°,每个不对称单位中含有1个AldBM066单体,马修斯系数为2.623/Da,溶剂含量约为53.02%.衍射数据的成功收集为解析巨大芽孢杆菌WSH-002中丙氨酸脱氢酶的三维结构奠定了前期基础,将有助于阐明以单体存在的丙氨酸脱氢酶的催化机制.
