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Abstract Bacillus subtilisis 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 inB. subtilis, here we report broad and efficient genetic code expansion inB. subtilisby 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 betweenE. coliandB. subtilisstop 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 inB. subtiliswill help uncover an abundance of biological insights and aid genetic code expansion in other organisms.
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The Role of Orthogonality in Genetic Code Expansion
The genetic code defines how information in the genome is translated into protein. Aside from a handful of isolated exceptions, this code is universal. Researchers have developed techniques to artificially expand the genetic code, repurposing codons and translational machinery to incorporate nonstandard amino acids (nsAAs) into proteins. A key challenge for robust genetic code expansion is orthogonality; the engineered machinery used to introduce nsAAs into proteins must co-exist with native translation and gene expression without cross-reactivity or pleiotropy. The issue of orthogonality manifests at several levels, including those of codons, ribosomes, aminoacyl-tRNA synthetases, tRNAs, and elongation factors. In this concept paper, we describe advances in genome recoding, translational engineering and associated challenges rooted in establishing orthogonality needed to expand the genetic code.
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- PAR ID:
- 10112261
- Date Published:
- Journal Name:
- Life
- Volume:
- 9
- Issue:
- 3
- ISSN:
- 2075-1729
- Page Range / eLocation ID:
- 58
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3390/life9030058
