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Designing efficient genetic code expansion in Bacillus subtilis to gain biological insights

View ORCID ProfileDevon A. Stork, View ORCID ProfileGeorgia R. Squyres, View ORCID ProfileErkin Kuru, View ORCID ProfileKatarzyna A. Gromek, View ORCID ProfileJonathan Rittichier, Aditya Jog, View ORCID ProfileBriana M. Burton, View ORCID ProfileGeorge M. Church, View ORCID ProfileEthan C. Garner, Aditya M. Kunjapur
doi: https://doi.org/10.1101/2021.02.19.432053
Devon A. Stork
1Department of Genetics, Harvard Medical School, Boston, MA 02157
2Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138
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Georgia R. Squyres
2Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138
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Erkin Kuru
1Department of Genetics, Harvard Medical School, Boston, MA 02157
3Wyss Institute for Biologically Inspired Engineering, Boston, MA 02115
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Katarzyna A. Gromek
4Department of Bacteriology, University of Wisconsin-Madison, Madison, WI 53706
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  • ORCID record for Katarzyna A. Gromek
Jonathan Rittichier
1Department of Genetics, Harvard Medical School, Boston, MA 02157
3Wyss Institute for Biologically Inspired Engineering, Boston, MA 02115
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Aditya Jog
1Department of Genetics, Harvard Medical School, Boston, MA 02157
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Briana M. Burton
4Department of Bacteriology, University of Wisconsin-Madison, Madison, WI 53706
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  • ORCID record for Briana M. Burton
George M. Church
1Department of Genetics, Harvard Medical School, Boston, MA 02157
3Wyss Institute for Biologically Inspired Engineering, Boston, MA 02115
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  • For correspondence: kunjapur@udel.edu egarner@g.harvard.edu gchurch@genetics.med.harvard.edu
Ethan C. Garner
2Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138
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  • For correspondence: kunjapur@udel.edu egarner@g.harvard.edu gchurch@genetics.med.harvard.edu
Aditya M. Kunjapur
1Department of Genetics, Harvard Medical School, Boston, MA 02157
5Department of Chemical and Biological Engineering, University of Delaware, Newark, DE 19716
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  • For correspondence: kunjapur@udel.edu egarner@g.harvard.edu gchurch@genetics.med.harvard.edu
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Abstract

Bacillus subtilis is a model Gram-positive bacterium, commonly used to explore questions across bacterial cell biology and for industrial uses. To enable greater understanding and control of proteins in B. subtilis, we demonstrate broad and efficient genetic code expansion in B. subtilis by incorporating 20 distinct non-standard amino acids within proteins using 3 different families of genetic code expansion systems and two choices of codons. We use these systems to achieve click-labelling, photo-crosslinking, and translational titration. These tools allow us to demonstrate differences between E. coli and B. subtilis stop codon suppression, validate a predicted protein-protein binding interface, and begin to interrogate properties underlying bacterial cytokinesis by precisely modulating cell division dynamics in vivo. We expect that the establishment of this simple and easily accessible chemical biology system in B. subtilis will help uncover an abundance of biological insights and aid genetic code expansion in other organisms.

Competing Interest Statement

G.M.C. has related financial interests in ReadCoor, EnEvolv, 64-X, and GRO Biosciences. For a complete list of G.M.C.s financial interests, please visit arep.med.harvard.edu/gmc/tech.html. No other authors have any conflict of interests.

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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 4.0 International license.
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Posted February 19, 2021.
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Designing efficient genetic code expansion in Bacillus subtilis to gain biological insights
Devon A. Stork, Georgia R. Squyres, Erkin Kuru, Katarzyna A. Gromek, Jonathan Rittichier, Aditya Jog, Briana M. Burton, George M. Church, Ethan C. Garner, Aditya M. Kunjapur
bioRxiv 2021.02.19.432053; doi: https://doi.org/10.1101/2021.02.19.432053
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Designing efficient genetic code expansion in Bacillus subtilis to gain biological insights
Devon A. Stork, Georgia R. Squyres, Erkin Kuru, Katarzyna A. Gromek, Jonathan Rittichier, Aditya Jog, Briana M. Burton, George M. Church, Ethan C. Garner, Aditya M. Kunjapur
bioRxiv 2021.02.19.432053; doi: https://doi.org/10.1101/2021.02.19.432053

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