Homogeneous expression of the PBAD promoter in Escherichia coli by constitutive expression of the low-affinity high-capacity AraE transporter
Homogeneous expression of the PBAD promoter in Escherichia coli by constitutive expression of the low-affinity high-capacity AraE transporter
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DOI：
10.1099/00221287-147-12-3241
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发表时间：
2001-12-01
期刊：
影响因子：
2.8
通讯作者：
Keasling, JD
中科院分区：
文献类型：
--
作者：
Khlebnikov, A;Datsenko, KA;Keasling, JD
置于大肠杆菌的阿拉伯糖可诱导的araBAD启动子(P-BAD)控制下的基因以全有或全无的方式表达，其中诱导细胞在群体中的百分比而不是单个细胞的诱导程度随着培养基中阿拉伯糖的浓度而变化。前人的工作表明，P-BAD基因的全或全不表达是由于编码低亲和力大容量转运蛋白(Arae)的基因依赖于阿拉伯糖的表达，而将P-BAD的异源基因在单个细胞中的表达可以通过将Arae基因置于不依赖于阿拉伯糖的启动子的控制下来调节。基于这些结果，开发了两个表达系统，以允许在P-BAD控制下对基因进行调控。在一个系统中，染色体上的天然Arae启动子被不同强度的构成启动子取代。在第二个系统中，将相同构成启动子控制下的arae基因放在一个中等拷贝的质粒上。这两个系统都允许可调控地表达质粒携带的P-BAD控制的异源基因，并允许在广泛的阿拉伯糖浓度范围内形成均一的细胞群体。虽然诱导度随组成启动子强度的不同而略有不同，但表达受阿拉伯糖浓度的影响最大。
Genes placed under the control of the arabinose-inducible araBAD promoter (P-BAD) of Escherichia coli are expressed in an all-or-none fashion, in which the percentage of induced cells in the population, rather than the degree of induction in individual cells, varies with the concentration of arabinose in the culture medium. Previous work showed that all-or-none gene expression from P-BAD was due to the arabinose-dependent expression of the gene encoding the low-affinity high-capacity transporter (araE), and that expression of heterologous genes from P-BAD in individual cells could be regulated by placing the araE gene under control of an arabinose-independent promoter. Based on these results, two expression systems were developed to allow regulatable control of genes under control of P-BAD. In one system, the native araE promoter on the chromosome was replaced by constitutive promoters of different strengths. In the second system, the araE gene under control of the same constitutive promoters was placed on a medium-copy plasmid. Both systems allow regulatable expression of a plasmid-borne P-BAD-controlled heterologous gene and a homogeneous population of cells over a wide range of arabinose concentrations. While the degree of induction varied slightly with the strength of the constitutive promoter, expression was affected most by the arabinose concentration.