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首页> 外文期刊>Microbiology >Self-replicating shuttle vectors based on pANS, a small endogenous plasmid of the unicellular cyanobacterium Synechococcus elongatus PCC 7942
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Self-replicating shuttle vectors based on pANS, a small endogenous plasmid of the unicellular cyanobacterium Synechococcus elongatus PCC 7942

机译：基于pANS的自复制穿梭载体，pANS是单细胞蓝藻长突触球菌PCC 7942的小内源质粒

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To facilitate development of synthetic biology tools for genetic engineering of cyanobacterial strains, we constructed pANS-derived self-replicating shuttle vectors that are based on the minimal replication element of the Synechococcus elongatus strain PCC 7942 plasmid pANS. To remove the possibility of homologous recombination events between the shuttle plasmids and the native pANS plasmid, the endogenous pANS was cured through plasmid incompatibility-mediated spontaneous loss. A heterologous toxin–antitoxin cassette was incorporated into the shuttle vectors for stable plasmid maintenance in the absence of antibiotic selection. The pANS-based shuttle vectors were shown to be able to carry a large 20 kb DNA fragment containing a gene cluster for biosynthesis of the omega-3 fatty acid eicosapentaenoic acid. Based on quantitative PCR analysis, there are about 10 copies of pANS and 3 copies of the large native plasmid pANL per chromosome in S. elongatus. Fluorescence levels of GFP reporter genes in a pANS-based vector were about 2.5-fold higher than when in pANL or integrated into the chromosome. In addition to its native host, pANS-based shuttle vectors were also found to replicate stably in the filamentous cyanobacterium Anabaena sp. strain PCC 7120. There were about 27 copies of a pANS-based shuttle vector, 9 copies of a pDU1-based shuttle vector and 3 copies of an RSF1010-based shuttle vector per genome when these three plasmids co-existed in Anabaena cells. The endogenous pANS from our S. elongatus laboratory strain was cloned in Escherichia coli, re-sequenced and re-annotated to update previously published sequencing data.
机译：为了促进对蓝细菌菌株进行基因工程的合成生物学工具的开发，我们构建了pANS衍生的自我复制穿梭载体，该载体基于细长拟乳球菌PCC 7942质粒pANS的最小复制元件。为了消除穿梭质粒和天然pANS质粒之间发生同源重组事件的可能性，可通过质粒不相容介导的自发丢失来治愈内源性pANS。在没有选择抗生素的情况下，将异源毒素-抗毒素盒整合到穿梭载体中，以稳定地维持质粒。基于pANS的穿梭载体显示出能够携带大的20 kb DNA片段，该片段包含用于生物合成ω-3脂肪酸二十碳五烯酸的基因簇。基于定量PCR分析，在延长链球菌中的每个染色体上存在约10个拷贝的pANS和3个拷贝的大天然质粒pANL。基于pANS的载体中GFP报告基因的荧光水平比在pANL中或整合到染色体中时高约2.5倍。除其天然宿主外，还发现基于pANS的穿梭载体在丝状蓝细菌Anabaena sp。中稳定复制。当这三个质粒共存在于Anabaena细胞中时，每个基因组大约有27个基于pANS的穿梭载体拷贝，9个基于pDU1的穿梭载体拷贝和3个基于RSF1010的穿梭载体拷贝。来自我们的S. elongatus实验室菌株的内源性pANS被克隆到大肠杆菌中，重新测序并重新注释以更新以前发布的测序数据。

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