A high-yield <i>Streptomyces</i> Transcription-translation toolkit for synthetic biology and natural product applications
Author(s)
Toh, Ming
Chengan, Kameshwari
Hanson, Tanith
Freemont, Paul S
Moore, Simon J
Type
Journal Article
Abstract
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.
Date Issued
2021-09-10
Date Acceptance
2021-09-01
Citation
Journal of Visualized Experiments, 2021, 2021 (175)
ISSN
1940-087X
Publisher
MyJove Corporation
Journal / Book Title
Journal of Visualized Experiments
Volume
2021
Issue
175
Copyright Statement
© 2021 JoVE.
Identifier
https://www.webofscience.com/api/gateway?GWVersion=2&SrcApp=PARTNER_APP&SrcAuth=LinksAMR&KeyUT=WOS:000708102100019&DestLinkType=FullRecord&DestApp=ALL_WOS&UsrCustomerID=a2bf6146997ec60c407a63945d4e92bb
Subjects
BIOSYNTHESIS
CELL
COMPLETE GENOME SEQUENCE
EXPRESSION
METABOLISM
Multidisciplinary Sciences
PROTEIN
Science & Technology
Science & Technology - Other Topics
Publication Status
Published
Coverage Spatial
United States
Article Number
ARTN e63012
Date Publish Online
2021-09-10