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Research

Biological systems - including the simplest cells - exhibit a broad range of functions to thrive in their environment. Research in the Imperial College Centre for Synthetic Biology is focused on the possibility of engineering the underlying biochemical processes to solve many of the challenges facing society, from healthcare to sustainable energy. In particular, we model, analyse, design and build biological and biochemical systems in living cells and/or in cell extracts, both exploring and enhancing the engineering potential of biology.

As part of our research we develop novel methods to accelerate the celebrated Design-Build-Test-Learn synthetic biology cycle. As such research in the Centre for Synthetic Biology highly multi- and interdisciplinary covering computational modelling and machine learning approaches; automated platform development and genetic circuit engineering ; multi-cellular and multi-organismal interactions, including gene drive and genome engineering; metabolic engineering; in vitro/cell-free synthetic biology; engineered phages and directed evolution; and biomimetics, biomaterials and biological engineering.

Publications

Citation

BibTex format

@article{Bubnov:2022:nar/gkac649,
author = {Bubnov, DM and Yuzbashev, TV and Khozov, AA and Melkina, OE and Vybornaya, TV and Stan, G-B and Sineoky, SP},
doi = {nar/gkac649},
journal = {Nucleic Acids Research},
title = {Robust counterselection and advanced λRed recombineering enable markerless chromosomal integration of large heterologous constructs.},
url = {http://dx.doi.org/10.1093/nar/gkac649},
volume = {50},
year = {2022}
}

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RIS format (EndNote, RefMan)

TY - JOUR
AB - Despite advances in bacterial genome engineering, delivery of large synthetic constructs remains challenging in practice. In this study, we propose a straightforward and robust approach for the markerless integration of DNA fragments encoding whole metabolic pathways into the genome. This approach relies on the replacement of a counterselection marker with cargo DNA cassettes via λRed recombineering. We employed a counterselection strategy involving a genetic circuit based on the CI repressor of λ phage. Our design ensures elimination of most spontaneous mutants, and thus provides a counterselection stringency close to the maximum possible. We improved the efficiency of integrating long PCR-generated cassettes by exploiting the Ocr antirestriction function of T7 phage, which completely prevents degradation of unmethylated DNA by restriction endonucleases in wild-type bacteria. The employment of highly restrictive counterselection and ocr-assisted λRed recombineering allowed markerless integration of operon-sized cassettes into arbitrary genomic loci of four enterobacterial species with an efficiency of 50-100%. In the case of Escherichia coli, our strategy ensures simple combination of markerless mutations in a single strain via P1 transduction. Overall, the proposed approach can serve as a general tool for synthetic biology and metabolic engineering in a range of bacterial hosts.
AU - Bubnov,DM
AU - Yuzbashev,TV
AU - Khozov,AA
AU - Melkina,OE
AU - Vybornaya,TV
AU - Stan,G-B
AU - Sineoky,SP
DO - nar/gkac649
PY - 2022///
SN - 0305-1048
TI - Robust counterselection and advanced λRed recombineering enable markerless chromosomal integration of large heterologous constructs.
T2 - Nucleic Acids Research
UR - http://dx.doi.org/10.1093/nar/gkac649
UR - https://www.ncbi.nlm.nih.gov/pubmed/35920321
UR - https://academic.oup.com/nar/advance-article/doi/10.1093/nar/gkac649/6654408
UR - http://hdl.handle.net/10044/1/98928
VL - 50
ER -