Skip to main content
bioRxiv
  • Home
  • About
  • Submit
  • ALERTS / RSS
Advanced Search
New Results

A Streptomyces venezuelae Cell-Free Toolkit for Synthetic Biology

View ORCID ProfileSimon J Moore, Hung-En Lai, Soo-Mei Chee, Ming Toh, Seth Coode, Patrick Capel, View ORCID ProfileChristophe Corre, View ORCID ProfileEmmanuel LC de los Santos, View ORCID ProfilePaul S Freemont
doi: https://doi.org/10.1101/2020.11.16.384693
Simon J Moore
1Centre for Synthetic Biology and Innovation, Imperial College London, South Kensington Campus, Exhibition Road, London, SW7 2AZ, UK
2Department Section of Structural and Synthetic Biology, Department of Infectious Disease; Imperial College London, South Kensington Campus, Exhibition Road, London, SW7 2AZ, UK
3School of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, UK
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Simon J Moore
  • For correspondence: S.J.R.Moore@kent.ac.uk p.freemont@imperial.ac.uk
Hung-En Lai
1Centre for Synthetic Biology and Innovation, Imperial College London, South Kensington Campus, Exhibition Road, London, SW7 2AZ, UK
2Department Section of Structural and Synthetic Biology, Department of Infectious Disease; Imperial College London, South Kensington Campus, Exhibition Road, London, SW7 2AZ, UK
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Soo-Mei Chee
2Department Section of Structural and Synthetic Biology, Department of Infectious Disease; Imperial College London, South Kensington Campus, Exhibition Road, London, SW7 2AZ, UK
5The London Biofoundry, Imperial College Translation & Innovation Hub, White City Campus, 80 Wood Lane, London W12 0BZ, UK
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Ming Toh
1Centre for Synthetic Biology and Innovation, Imperial College London, South Kensington Campus, Exhibition Road, London, SW7 2AZ, UK
2Department Section of Structural and Synthetic Biology, Department of Infectious Disease; Imperial College London, South Kensington Campus, Exhibition Road, London, SW7 2AZ, UK
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Seth Coode
3School of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, UK
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Patrick Capel
4Warwick Integrative Synthetic Biology Centre, School of Life Sciences, University of Warwick, Gibbet Hill Road, Coventry, CV4 7AL, UK
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Christophe Corre
4Warwick Integrative Synthetic Biology Centre, School of Life Sciences, University of Warwick, Gibbet Hill Road, Coventry, CV4 7AL, UK
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Christophe Corre
Emmanuel LC de los Santos
4Warwick Integrative Synthetic Biology Centre, School of Life Sciences, University of Warwick, Gibbet Hill Road, Coventry, CV4 7AL, UK
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Emmanuel LC de los Santos
Paul S Freemont
1Centre for Synthetic Biology and Innovation, Imperial College London, South Kensington Campus, Exhibition Road, London, SW7 2AZ, UK
2Department Section of Structural and Synthetic Biology, Department of Infectious Disease; Imperial College London, South Kensington Campus, Exhibition Road, London, SW7 2AZ, UK
5The London Biofoundry, Imperial College Translation & Innovation Hub, White City Campus, 80 Wood Lane, London W12 0BZ, UK
6UK Dementia Research Institute Care Research and Technology Centre, Imperial College London, Hammersmith Campus, Du Cane Road, London, W12 0N
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Paul S Freemont
  • For correspondence: S.J.R.Moore@kent.ac.uk p.freemont@imperial.ac.uk
  • Abstract
  • Full Text
  • Info/History
  • Metrics
  • Preview PDF
Loading

Abstract

Prokaryotic cell-free coupled transcription-translation (TX-TL) systems are emerging as a powerful tool to examine natural product biosynthetic pathways in a test-tube. The key advantages of this approach are the reduced experimental timescales and controlled reaction conditions. In order to realise this potential, specialised cell-free systems in organisms enriched for biosynthetic gene clusters, with strong protein production and well-characterised synthetic biology tools, is essential. The Streptomyces genus is a major source of natural products. To study enzymes and pathways from Streptomyces, we originally developed a homologous Streptomyces cell-free system to provide a native protein folding environment, a high G+C (%) tRNA pool and an active background metabolism. However, our initial yields were low (36 μg/mL) and showed a high level of batch-to-batch variation. Here, we present an updated high-yield and robust Streptomyces TX-TL protocol, reaching up to yields of 266 μg/mL of expressed recombinant protein. To complement this, we rapidly characterise a range of DNA parts with different reporters, express high G+C (%) biosynthetic genes and demonstrate an initial proof of concept for combined transcription, translation and biosynthesis of Streptomyces metabolic pathways in a single ‘one-pot’ reaction.

Competing Interest Statement

The authors have declared no competing interest.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
Back to top
PreviousNext
Posted November 16, 2020.
Download PDF
Email

Thank you for your interest in spreading the word about bioRxiv.

NOTE: Your email address is requested solely to identify you as the sender of this article.

Enter multiple addresses on separate lines or separate them with commas.
A Streptomyces venezuelae Cell-Free Toolkit for Synthetic Biology
(Your Name) has forwarded a page to you from bioRxiv
(Your Name) thought you would like to see this page from the bioRxiv website.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Share
A Streptomyces venezuelae Cell-Free Toolkit for Synthetic Biology
Simon J Moore, Hung-En Lai, Soo-Mei Chee, Ming Toh, Seth Coode, Patrick Capel, Christophe Corre, Emmanuel LC de los Santos, Paul S Freemont
bioRxiv 2020.11.16.384693; doi: https://doi.org/10.1101/2020.11.16.384693
Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
Citation Tools
A Streptomyces venezuelae Cell-Free Toolkit for Synthetic Biology
Simon J Moore, Hung-En Lai, Soo-Mei Chee, Ming Toh, Seth Coode, Patrick Capel, Christophe Corre, Emmanuel LC de los Santos, Paul S Freemont
bioRxiv 2020.11.16.384693; doi: https://doi.org/10.1101/2020.11.16.384693

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
  • Tweet Widget
  • Facebook Like
  • Google Plus One

Subject Area

  • Synthetic Biology
Subject Areas
All Articles
  • Animal Behavior and Cognition (2633)
  • Biochemistry (5221)
  • Bioengineering (3643)
  • Bioinformatics (15709)
  • Biophysics (7213)
  • Cancer Biology (5592)
  • Cell Biology (8042)
  • Clinical Trials (138)
  • Developmental Biology (4732)
  • Ecology (7458)
  • Epidemiology (2059)
  • Evolutionary Biology (10518)
  • Genetics (7697)
  • Genomics (10080)
  • Immunology (5145)
  • Microbiology (13823)
  • Molecular Biology (5354)
  • Neuroscience (30574)
  • Paleontology (211)
  • Pathology (870)
  • Pharmacology and Toxicology (1519)
  • Physiology (2233)
  • Plant Biology (4981)
  • Scientific Communication and Education (1036)
  • Synthetic Biology (1379)
  • Systems Biology (4129)
  • Zoology (803)