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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.