Regulatable arabinose-inducible gene expression system with consistent control in all cells of a culture
Regulatable arabinose-inducible gene expression system with consistent control in all cells of a culture
复制标题
DOI：
10.1128/jb.182.24.7029-7034.2000
复制
发表时间：
2000-12-01
影响因子：
3.2
通讯作者：
Keasling, JD
中科院分区：
文献类型：
--
作者：
Khlebnikov, A;Risa, O;Keasling, JD
阿拉伯糖诱导型启动子P-BAD受到全或无诱导，其中中等浓度的阿拉伯糖产生完全诱导和未诱导的细胞亚群。为了在同质细胞群中构建具有可调控控制的宿主载体表达系统，将大肠杆菌的araE基因克隆到RSF 1010衍生的质粒中，该质粒在异丙基-β-D-硫代半乳糖苷诱导型P-tac和P-taclac启动子的控制下。该基因编码低亲和力、高容量的阿拉伯糖转运蛋白，并由阿拉伯糖诱导型启动子天然控制。为了检测阿拉伯糖非依赖性araE表达对群体同质性和细胞特异性表达的影响，将gfpuv基因置于pMB 1衍生的质粒pBAD 24上的阿拉伯糖诱导型araBAD启动子（P-BAD）的控制下。将转运蛋白和报告质粒转化到具有天然阿拉伯糖转运系统的大肠杆菌菌株和一种或两种阿拉伯糖转运系统（araE和/或araFGH）缺陷的菌株中。使用流式细胞术在这些菌株中研究了阿拉伯糖浓度和阿拉伯糖非依赖性转运控制对群体均一性的影响。在所有诱导剂浓度下，在整个群体中均匀诱导携带转运蛋白和报告质粒的araE和araE araFGH突变株，并且个体细胞中的基因表达水平随阿拉伯糖浓度而变化。相反，表达天然araE和araFGH基因并携带转运蛋白和报告质粒的亲本菌株表现出全或无行为。这项工作证明了包括转运基因的重要性，该转运基因独立于诱导剂进行控制，以在培养物的所有细胞中实现可调控和一致的诱导。
The arabinose-inducible promoter P-BAD is subject to all-or-none induction, in which intermediate concentrations of arabinose give rise to subpopulations of cells that are fully induced and uninduced. To construct a host-vector expression system with regulatable control in a homogeneous population of cells, the araE gene of Escherichia coli was cloned into an RSF1010-derived plasmid under control of the isopropyl-beta -D-thiogalactopyranoside-inducible P-tac and P-taclac promoters. This gene encodes the low-affinity, high-capacity arabinose transport protein and is controlled natively by an arabinose-inducible promoter. To detect the effect of arabinose-independent araE expression on population homogeneity and cell-specific expression, the gfpuv gene was placed under control of the arabinose-inducible araBAD promoter (P-BAD) on the pMB1-derived plasmid pBAD24. The transporter and reporter plasmids were transformed into E, coli strains with native arabinose transport systems and strains deficient in one or both of the arabinose transport systems (araE and/or araFGH). The effects of the arabinose concentration and arabinose-independent transport control on population homogeneity were investigated in these strains using flow cytometry. The araE, and araE araFGH mutant strains harboring the transporter and reporter plasmids were uniformly induced across the population at all inducer concentrations, and the level of gene expression in individual cells varied with arabinose concentration, In contrast, the parent strain, which expressed the native araE and araFGH genes and harbored the transporter and reporter plasmids, exhibited all-or-none behavior. This work demonstrates the importance of including a transport gene that is controlled independently of the inducer to achieve regulatable and consistent induction in all cells of the culture.