Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Creating custom synthetic genomes in Escherichia coli with REXER and GENESIS

Nature Protocols volume 16, pages 2345–2380 (2021)Cite this article

Subjects

Abstract

We previously developed REXER (Replicon EXcision Enhanced Recombination); this method enables the replacement of >100 kb of the Escherichia coli genome with synthetic DNA in a single step and allows the rapid identification of non-viable or otherwise problematic sequences with nucleotide resolution. Iterative repetition of REXER (GENESIS, GENomE Stepwise Interchange Synthesis) enables stepwise replacement of longer contiguous sections of genomic DNA with synthetic DNA, and even the replacement of the entire E. coli genome with synthetic DNA. Here we detail protocols for REXER and GENESIS. A standard REXER protocol typically takes 7–10 days to complete. Our description encompasses (i) synthetic DNA design, (ii) assembly of synthetic DNA constructs, (iii) utilization of CRISPR–Cas9 coupled to lambda-red recombination and positive/negative selection to enable the high-fidelity replacement of genomic DNA with synthetic DNA (or insertion of synthetic DNA), (iv) evaluation of the success of the integration and replacement and (v) identification of non-tolerated synthetic DNA sequences with nucleotide resolution. This protocol provides a set of precise genome engineering methods to create custom synthetic E. coli genomes.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Schematic of REXER and GENESIS.
Fig. 2: BAC assembly and delivery.
Fig. 3: Components for REXER.
Fig. 4: REXER workflow.
Fig. 5: Identifying deleterious sequences in synthetic DNA.
Fig. 6: Testing potential fixes for deleterious synthetic sequences.

Similar content being viewed by others

Data availability

No new data were generated or analyzed while preparing this protocol. Representative data are available in refs. 3,14.

Code availability

Code used for next-generation sequencing analysis is freely available at https://github.com/TiongSun/iSeq.

References

  1. Gibson, D. G. et al. Creation of a bacterial cell controlled by a chemically synthesized genome. Science 329, 52–56 (2010).

    Article  CAS  Google Scholar 

  2. Hutchison, C. A. 3rd et al. Design and synthesis of a minimal bacterial genome. Science 351, aad6253 (2016).

    Article  Google Scholar 

  3. Fredens, J. et al. Total synthesis of Escherichia coli with a recoded genome. Nature 569, 514–518 (2019).

    Article  CAS  Google Scholar 

  4. Richardson, S. M. et al. Design of a synthetic yeast genome. Science 355, 1040 (2017).

    Article  CAS  Google Scholar 

  5. Venetz, J. E. et al. Chemical synthesis rewriting of a bacterial genome to achieve design flexibility and biological functionality. Proc. Natl. Acad. Sci. USA 116, 8070 (2019).

    Article  CAS  Google Scholar 

  6. Krishnakumar, R. et al. Simultaneous non-contiguous deletions using large synthetic DNA and site-specific recombinases. Nucleic Acids Res. 42, e111–e111 (2014).

    Article  Google Scholar 

  7. Santos, C. N., Regitsky, D. D. & Yoshikuni, Y. Implementation of stable and complex biological systems through recombinase-assisted genome engineering. Nat. Commun. 4, 2503 (2013).

    Article  Google Scholar 

  8. Santos, C. N. & Yoshikuni, Y. Engineering complex biological systems in bacteria through recombinase-assisted genome engineering. Nat. Protoc. 9, 1320–1336 (2014).

    Article  CAS  Google Scholar 

  9. Wang, G. et al. CRAGE enables rapid activation of biosynthetic gene clusters in undomesticated bacteria. Nat. Microbiol. 4, 2498–2510 (2019).

    Article  Google Scholar 

  10. Datsenko, K. A. & Wanner, B. L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. USA 97, 6640 (2000).

    Article  CAS  Google Scholar 

  11. Wannier, T. M. et al. Improved bacterial recombineering by parallelized protein discovery. Proc. Natl. Acad. Sci. USA 117, 13689 (2020).

    Article  CAS  Google Scholar 

  12. Zhang, Y., Buchholz, F., Muyrers, J. P. P. & Stewart, A. F. A new logic for DNA engineering using recombination in Escherichia coli. Nat. Genet. 20, 123–128 (1998).

    Article  CAS  Google Scholar 

  13. Lau, Y. H. et al. Large-scale recoding of a bacterial genome by iterative recombineering of synthetic DNA. Nucleic Acids Res. 45, 6971–6980 (2017).

    Article  CAS  Google Scholar 

  14. Wang, K. et al. Defining synonymous codon compression schemes by genome recoding. Nature 539, 59–64 (2016).

    Article  CAS  Google Scholar 

  15. Kouprina, N. & Larionov, V. Selective isolation of genomic loci from complex genomes by transformation-associated recombination cloning in the yeast Saccharomyces cerevisiae. Nat. Protoc. 3, 371–377 (2008).

    Article  CAS  Google Scholar 

  16. Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012).

    Article  CAS  Google Scholar 

  17. Li, H. et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).

    Article  Google Scholar 

  18. Thorvaldsdottir, H., Robinson, J. T. & Mesirov, J. P. Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration. Brief Bioinform. 14, 178–192 (2013).

    Article  CAS  Google Scholar 

  19. Mosberg, J. A., Lajoie, M. J. & Church, G. M. Lambda red recombineering in Escherichia coli occurs through a fully single-stranded intermediate. Genetics 186, 791–799 (2010).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by the Medical Research Council (MRC), UK (MC_U105181009 and MC_UP_A024_1008), the Medical Research Foundation (MRF-109-0003-RG-CHIN/C0741) and an ERC Advanced Grant SGCR, all to J.W.C., and by the Lundbeck Foundation (R232-2016-3474) to J.F. J.W.C. thanks H. Pelham for supporting this project. We are grateful to the LMB Media Kitchen for help with preparing materials.

Author information

Author notes

  1. These authors contributed equally: Wesley E. Robertson, Louise F. H. Funke, Daniel de la Torre, Julius Fredens.

Authors and Affiliations

  1. Medical Research Council Laboratory of Molecular Biology, Cambridge, England, UK

    Wesley E. Robertson, Louise F. H. Funke, Daniel de la Torre, Julius Fredens & Jason W. Chin

  2. Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA

    Kaihang Wang

Authors
  1. Wesley E. Robertson
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Louise F. H. Funke
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Daniel de la Torre
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Julius Fredens
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Kaihang Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jason W. Chin
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

All authors contributed to developing this protocol and writing this paper. J.W.C. supervised the project.

Corresponding authors

Correspondence to Kaihang Wang or Jason W. Chin.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Protocols thanks Byung-Kwan Cho, Bogumil Karas and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

Key references using this protocol

Wang, K. et al. Nature 539, 59–64 (2016): https://doi.org/10.1038/nature20124

Fredens, J. et al. Nature 569, 514–518 (2019): https://doi.org/10.1038/s41586-019-1192-5

Supplementary information

Supplementary Information

Supplementary Fig. 1.

Reporting Summary

Supplementary Table 1

Primers used in this study.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Robertson, W.E., Funke, L.F.H., de la Torre, D. et al. Creating custom synthetic genomes in Escherichia coli with REXER and GENESIS. Nat Protoc 16, 2345–2380 (2021). https://doi.org/10.1038/s41596-020-00464-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41596-020-00464-3

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing