Overcoming the Challenges of Megabase-Sized Plasmid Construction in Escherichia coli
Overcoming the Challenges of Megabase-Sized Plasmid Construction in Escherichia coli
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DOI：
10.1021/acssynbio.0c00008
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发表时间：
2020-06-19
影响因子：
4.7
通讯作者：
Su'etsugu, Masayuki
中科院分区：
文献类型：
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作者：
Mukai, Takahito;Yoneji, Tatsuya;Su'etsugu, Masayuki
虽然大肠杆菌是质粒构建的常用工具，但这种细菌被认为“不适合”构建大小超过500个内切酶的大质粒。我们假设传统的质粒载体可能缺乏稳定复制和分离这样一个大质粒所需的一些调控DNA元件。此外，使用一些位点特异性重组系统可以促进大DNA片段的克隆。在这里，我们展示了两种策略，构建1兆碱基（1-Mb）的次级染色体，通过使用新的细菌人工染色体（BAC）载体。首先，对基因组缩减的E. coli菌株的染色体分裂为2-Mb和1-Mb两条染色体，其中较小的一条染色体具有管氏弧菌次级染色体的复制起点和分配位点。这种染色体分裂方法（Flp-POP克隆）通过翻转酶介导的切除起作用，这与分裂的氯霉素抗性基因的重新组装相一致，从而允许氯霉素选择。接下来，我们开发了一种新的供体方法（oriT-POP克隆）和一个完全装备的BAC载体（pMegaBAC 1H），用于开发1-Mb质粒。通过接合将两个0.5-Mb基因组区域依次从两个供体菌株转移到受体菌株，并在受体菌株中被pMegaBAC 1H捕获以产生1-Mb质粒。该质粒可传递到另一株E.大肠杆菌菌株。此外，这些1-Mb的次级染色体可在体外用重组E.大肠杆菌染色体复制循环反应（RCR）。这些策略和技术将使E.大肠杆菌细胞是设计染色体工程的生产工厂。
Although Escherichia coli has been a popular tool for plasmid construction, this bacterium was believed to be "unsuitable" for constructing a large plasmid whose size exceeds 500 kilobases. We assumed that traditional plasmid vectors may lack some regulatory DNA elements required for the stable replication and segregation of such a large plasmid. In addition, the use of a few site-specific recombination systems may facilitate cloning of large DNA segments. Here we show two strategies for constructing 1-megabase (1-Mb) secondary chromosomes by using new bacterial artificial chromosome (BAC) vectors. First, the 3-Mb genome of a genome-reduced E. coli strain was split into two chromosomes (2-Mb and 1-Mb), of which the smaller one has the origin of replication and the partitioning locus of the Vibrio tubiashii secondary chromosome. This chromosome fission method (Flp-POP cloning) works via flippase-mediated excision, which coincides with the reassembly of a split chloramphenicol resistance gene, allowing chloramphenicol selection. Next, we developed a new doning method (oriT-POP cloning) and a fully equipped BAC vector (pMegaBAC1H) for developing a 1-Mb plasmid. Two 0.5-Mb genomic regions were sequentially transferred from two donor strains to a recipient strain via conjugation and captured by pMegaBAC1H in the recipient strain to produce a 1-Mb plasmid. This 1-Mb plasmid was transmissible to another E. coli strain via conjugation. Furthermore, these 1-Mb secondary chromosomes were amplifiable in vitro by using the reconstituted E. coli chromosome replication cycle reaction (RCR). These strategies and technologies would make popular E. coli cells a productive factory for designer chromosome engineering.