A high-throughput, restriction-free cloning and screening strategy based on ccdB-gene replacement.
A high-throughput, restriction-free cloning and screening strategy based on ccdB-gene replacement.
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DOI：
10.1186/1475-2859-13-38
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发表时间：
2014-03-10
影响因子：
6.4
通讯作者：
Bjerga GE
中科院分区：
文献类型：
--
作者：
Lund BA;Leiros HK;Bjerga GE
In high-throughput demanding fields, such as biotechnology and structural biology, molecular cloning is an essential tool in obtaining high yields of recombinant protein. Here, we address recently developed restriction-free methods in cloning, and present a more cost-efficient protocol that has been optimized to improve both cloning and clone screening. In our case study, three homologous β-lactamase genes were successfully cloned using these restriction-free protocols. To clone the genes, we chose a gene replacement strategy, where the recombinant genes contained overhangs that targeted a region of the expression vector including a cytotoxin-encoding ccdB-gene. We provide further evidence that gene replacement can be applied with high-throughput cloning protocols. Targeting a replacement of the ccdB-gene was found to be very successful for counterselection using these protocols. This eliminated the need for treatment with the restriction enzyme DpnI that has so far been the preferred clone selection approach. We thus present an optimized cloning protocol using a restriction-free ccdB-gene replacement strategy, which allows for parallel cloning at a high-throughput level.
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