Incorporation of a Chemically Diverse Set of Non-Standard Amino Acids into a Gram-Positive Organism

Devon A Stork; Michaela A Jones; Ethan C Garner; Aditya M Kunjapur

doi:10.21769/BioProtoc.4507

Incorporation of a Chemically Diverse Set of Non-Standard Amino Acids into a Gram-Positive Organism

Bio Protoc. 2022 Sep 5;12(17):e4507. doi: 10.21769/BioProtoc.4507.

Authors

Devon A Stork^{1

2}, Michaela A Jones³, Ethan C Garner², Aditya M Kunjapur³

Affiliations

¹ Department of Genetics, Harvard Medical School, Boston, MA, USA.
² Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA, USA.
³ Department of Chemical and Biological Engineering, University of Delaware, Newark, DE, USA.

Abstract

The incorporation of non-standard amino acids (nsAAs) within proteins and peptides through genetic code expansion introduces novel chemical functionalities such as photo-crosslinking and bioconjugation. Given the utility of Bacillus subtilis in fundamental and applied science, we extended existing nsAA incorporation technology from Escherichia coli into B. subtilis , demonstrating incorporation of 20 unique nsAAs. The nsAAs we succeeded in incorporating within proteins conferred properties that included fluorescence, photo-crosslinking, and metal chelation. Here, we describe the reagents, equipment, and protocols to test for nsAA incorporation at a small scale (96-well plate and culture tube scales). We report specific media requirements for certain nsAAs, including two variants for different media conditions. Our protocol provides a consistent and reproducible method for incorporation of a chemically diverse set of nsAAs into a model Gram-positive organism.

Keywords: Bacillus subtilis; Genetic code expansion; Non-standard amino acid; Noncanonical amino acid; Synthetic biology; Translational control; Unnatural amino acid.