A novel DMAPP-responding genetic circuit sensor for high-throughput screening and evolving isoprene synthase
A novel DMAPP-responding genetic circuit sensor for high-throughput screening and evolving isoprene synthase
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一种新型 DMAPP 响应遗传电路传感器，用于高通量筛选和进化异戊二烯合酶
DOI：
10.1007/s00253-017-8676-8
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发表时间：
2018-02-01
影响因子：
5
通讯作者：
Tan, Tian-Wei
中科院分区：
文献类型：
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作者：
Liu, Chun-Li;Cai, Jing-Yi;Tan, Tian-Wei
高通量筛选是整理生物数据的流行工具，否则需要使用过多的资源。本研究基于修饰的L-阿拉伯糖操纵子构建了响应二甲基烯丙基二磷酸（DMAPP）的人工遗传电路传感器，用于大肠杆菌（E. coli）的高通量筛选和异戊二烯合酶（ispS）进化。第一步，用 anispSgene 替换 L-阿拉伯糖配体结合域 (LBD) 的 DNA 序列，以使 AraC 操纵子能够响应 DMAPP（IspS 酶的底物）。然后，还引入了增强型 GFP (eGFP) 作为 pBAD 启动子的报告基因。使用两种工具之一监测报告基因的表达水平：流式细胞仪（FCM）和酶标仪。随后，我们观察到高DMAPP浓度导致低eGFP荧光，并且消耗DMAPP的ispS基因的过表达导致高eGFP表达。这些结果表明，人工遗传电路传感器直接响应细胞内DMAPP浓度，并且IspS酶的表达可以与eGFP的表达水平正相关。最后，我们从anispSgene文库中鉴定出两个具有不同活性的IspS突变体，并进一步验证了筛选方法。
High-throughput screening is a popular tool for collating biological data which would otherwise require the use of excessive resources. In this study, an artificial genetic circuit sensor responding to dimethylallyl diphosphate (DMAPP) was constructed based on a modified L-arabinose operon for high-throughput screening and isoprene synthase (ispS) evolution inEscherichia coli(E. coli). As a first step, the DNA sequence of the L-arabinose ligand-binding domain (LBD) was replaced with anispSgene to enable the AraC operon responding to DMAPP, which is the substrate of the IspS enzyme. Then, an enhanced GFP (eGFP) was also introduced as a reporter for pBAD promoter. The expression level of the reporter was monitored using either of the two tools: flow cytometer (FCM) and microplate reader. Sequentially, we observed that a high DMAPP concentration led to low eGFP fluorescence, and the overexpression ofispSgene, which consumes DMAPP, resulted in a high eGFP expression. These results demonstrated that the artificial genetic circuit sensor responded directly to the intracellular concentration of DMAPP, and the expression of IspS enzyme could be positively correlated to the expression level of eGFP. Finally, we identified two IspS mutants with different activities from anispSgene library and further validated the screening method.