Substrate-induced gene expression (SIGEX) screening of metagenome libraries
Substrate-induced gene expression (SIGEX) screening of metagenome libraries
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DOI：
10.1038/nprot.2008.96
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发表时间：
2008-01-01
期刊：
影响因子：
14.8
通讯作者：
Watanabe, Kazuya
中科院分区：
文献类型：
--
作者：
Uchiyama, Taku;Watanabe, Kazuya
底物诱导的基因表达筛选（SIGEX）已被开发用于从环境宏基因组中分离新的分解代谢基因，特别是使用常规基因克隆方法难以获得的基因。在SIGEX中，将限制酶消化的宏基因组片段连接到操纵子捕获载体（例如，p18GFP），并通过转化克隆宿主（例如，大肠杆菌）。对文库进行底物依赖性基因诱导测定，并通过检测共表达标记物（例如，GFP）。如果使用荧光激活细胞分选（FACS）来选择GFP表达细胞，则高通量筛选是可能的。上述SIGEX程序需要类似于17 d。在该协议中，一个广泛适用的SIGEX计划，沿着与典型的实验结果。
Substrate-induced gene-expression screening (SIGEX) has been developed for isolating novel catabolic genes from environmental metagenomes, particularly genes that are difficult to obtain using conventional gene-cloning methods. In SIGEX, restriction enzyme-digested metagenome fragments are ligated into an operon-trap vector (e.g., p18GFP), and a library is constructed in a liquid culture by transforming a cloning host (e.g., Escherichia coli). The library is subjected to a substrate-dependent gene-induction assay, and positive cells are selected by detecting activity of a co-expressed marker (e.g., GFP) encoded in the vector. High-throughput screening is possible if fluorescence-activated cell sorting (FACS) is used to select GFP-expressing cells. The abovementioned SIGEX procedure requires similar to 17 d. In this protocol, a widely applicable SIGEX scheme is presented along with typical experimental results.