CyanoGate: A Modular Cloning Suite for Engineering Cyanobacteria Based on the Plant MoClo Syntax
CyanoGate: A Modular Cloning Suite for Engineering Cyanobacteria Based on the Plant MoClo Syntax
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DOI：
10.1104/pp.18.01401
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发表时间：
2019-05-01
期刊：
影响因子：
7.4
通讯作者：
McCormick, Alistair J.
中科院分区：
文献类型：
--
作者：
Vasudevan, Ravendran;Gale, Grant A. R.;McCormick, Alistair J.
合成生物学研究的最新进展得到了可用基因组信息指数增长和先进DNA组装工具激增的支持。质粒载体组装标准和部件库的采用大大提高了研究的可重复性以及不同实验室和生物系统之间的部件交换。然而，一个标准化的模块化克隆（MoClo）系统尚未提供蓝藻，落后于其他原核生物在合成生物学，尽管他们的巨大潜力，生物技术应用。通过构建Plant Golden Gate MoClo套件的组装库和语法，我们开发了一个名为CyanoGate的多功能系统，将蓝藻与植物和藻类系统结合起来。在这里，我们描述了一套部件和受体载体的产生，用于（1）使用整合受体载体进行标记/未标记敲除或整合，以及（2）使用已知和以前未描述的复制载体进行瞬时多基因表达和阻遏系统。我们测试和比较了CyanoGate系统中建立的模型蓝藻集胞藻属PCC 6803和最近描述的快速生长菌株细长聚球藻UTEX 2973。UTEX 2973快速生长表型仅在特定生长条件下明显;然而，UTEX 2973积累了高水平的具有强天然或合成启动子的蛋白质。该系统是公开可用的，可以很容易地扩展到容纳其他标准化的MoClo部件，以加速为蓝藻群落开发可靠的合成生物学工具。
Recent advances in synthetic biology research have been underpinned by an exponential increase in available genomic information and a proliferation of advanced DNA assembly tools. The adoption of plasmid vector assembly standards and parts libraries has greatly enhanced the reproducibility of research and the exchange of parts between different labs and biological systems. However, a standardized modular cloning (MoClo) system is not yet available for cyanobacteria, which lag behind other prokaryotes in synthetic biology despite their huge potential regarding biotechnological applications. By building on the assembly library and syntax of the Plant Golden Gate MoClo kit, we have developed a versatile system called CyanoGate that unites cyanobacteria with plant and algal systems. Here, we describe the generation of a suite of parts and acceptor vectors for making (1) marked/unmarked knock-outs or integrations using an integrative acceptor vector, and (2) transient multigene expression and repression systems using known and previously undescribed replicative vectors. We tested and compared the CyanoGate system in the established model cyanobacterium Synechocystis sp. PCC 6803 and the more recently described fast-growing strain Synechococcus elongatus UTEX 2973. The UTEX 2973 fast-growth phenotype was only evident under specific growth conditions; however, UTEX 2973 accumulated high levels of proteins with strong native or synthetic promoters. The system is publicly available and can be readily expanded to accommodate other standardized MoClo parts to accelerate the development of reliable synthetic biology tools for the cyanobacterial community.