Toh Ming, Chengan Kameshwari, Hanson Tanith, Freemont Paul S, Moore Simon J
Centre for Synthetic Biology and Innovation, South Kensington Campus; Department of Medicine, South Kensington Campus; Section of Structural and Synthetic Biology, Department of Infectious Disease, Imperial College London; Sir Alexander Fleming Building, South Kensington Campus.
School of Biosciences, Division of Natural Sciences, University of Kent.
J Vis Exp. 2021 Sep 10(175). doi: 10.3791/63012.
Streptomyces spp. are a major source of clinical antibiotics and industrial chemicals. Streptomyces venezuelae ATCC 10712 is a fast-growing strain and a natural producer of chloramphenicol, jadomycin, and pikromycin, which makes it an attractive candidate as a next-generation synthetic biology chassis. Therefore, genetic tools that accelerate the development of S. venezuelae ATCC 10712, as well as other Streptomyces spp. models, are highly desirable for natural product engineering and discovery. To this end, a dedicated S. venezuelae ATCC 10712 cell-free system is provided in this protocol to enable high-yield heterologous expression of high G+C (%) genes. This protocol is suitable for small-scale (10-100 μL) batch reactions in either 96-well or 384-well plate format, while reactions are potentially scalable. The cell-free system is robust and can achieve high yields (~5-10 μM) for a range of recombinant proteins in a minimal setup. This work also incorporates a broad plasmid toolset for real-time measurement of mRNA and protein synthesis, as well as in-gel fluorescence staining of tagged proteins. This protocol can also be integrated with high-throughput gene expression characterization workflows or the study of enzyme pathways from high G+C (%) genes present in Actinomycetes genomes.
链霉菌属是临床抗生素和工业化学品的主要来源。委内瑞拉链霉菌ATCC 10712是一种生长迅速的菌株，是氯霉素、制霉素和匹克霉素的天然生产者，这使其成为下一代合成生物学底盘的有吸引力的候选者。因此，加速委内瑞拉链霉菌ATCC 10712以及其他链霉菌属模型开发的遗传工具对于天然产物工程和发现来说是非常需要的。为此，本方案提供了一个专门的委内瑞拉链霉菌ATCC 10712无细胞系统，以实现高G+C（%）基因的高产异源表达。本方案适用于96孔或384孔板形式的小规模（10-100 μL）分批反应，而反应具有潜在的可扩展性。该无细胞系统稳健，在最小设置下可实现一系列重组蛋白的高产率（~5-10 μM）。这项工作还纳入了一个广泛的质粒工具集，用于实时测量mRNA和蛋白质合成，以及标记蛋白的凝胶内荧光染色。本方案还可与高通量基因表达表征工作流程或放线菌基因组中高G+C（%）基因的酶途径研究相结合。
