Delivery of novel replicating vectors to Synechococcus sp. PCC 7002 via natural transformation of plasmid multimers

Cody Kamoku, Cheyanna Cooper, Ashley Straub, Nathan Miller, David R Nielsen
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Abstract

In most cyanobacteria, genetic engineering efforts currently rely upon chromosomal integration; a time-consuming process due to their polyploid nature. To enhance strain construction, here we develop and characterize two novel replicating plasmids for use in Synechococcus sp. PCC 7002. Following an initial screen of plasmids comprising seven different origins of replication, two were found capable of replication: one based on the WVO1 broad host range plasmid and the other a shuttle vector derived from pCB2.4 from Synechocystis sp. PCC 6803. These were then used to construct a set of new replicating plasmids, which were shown to be both co-transformable and stably maintained in PCC 7002 at copy numbers between 0.6-1.4 and 7-16, respectively. Lastly, we demonstrate the importance of using multimeric plasmids during natural transformation of PCC 7002, with higher order multimers providing a 30-fold increase in transformation efficiency relative to monomeric plasmids. Useful considerations and methods for enhancing multimer content in plasmid samples are also presented.
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通过质粒多聚体的自然转化向 Synechococcus sp.
在大多数蓝藻中,基因工程工作目前依赖于染色体整合;由于蓝藻的多倍体性质,这一过程非常耗时。为了加强菌株构建,我们在此开发并鉴定了两种新型复制质粒,用于 Synechococcus sp.在对包含七种不同复制起源的质粒进行初步筛选后,我们发现了两种能够复制的质粒:一种基于 WVO1 广宿主范围质粒,另一种是来自 Synechocystis sp.PCC 6803 的 pCB2.4 的穿梭载体。随后,我们利用这些质粒构建了一组新的复制质粒,结果表明,这些质粒在 PCC 7002 中既能共转化,又能稳定维持,拷贝数分别在 0.6-1.4 和 7-16 之间。最后,我们证明了在 PCC 7002 的自然转化过程中使用多聚质粒的重要性,与单体质粒相比,高阶多聚质粒的转化效率提高了 30 倍。我们还介绍了提高质粒样品中多聚体含量的有用注意事项和方法。
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