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Cross species expression of the T7 RNAP and TetR also required rational design of their ribosome binding sites and protein coding sequences. We employed multi objective codon optimization to identify synonymous codons that would satisfy three criteria: maximizing translation elongation rates across both Gram negative and Gram positive bacteria, using Escherichia coli DH10B, Pseudomonas putida KT2440 and Bacillus subtilis 168 as examples; eliminating direct or inverted repeat sequences that promote genetic instability; and removing putative RNAse E and RNAse III binding sites to improve mRNA stability34,35,36,37,38. To control their expression levels in different bacterial hosts, we then employed the RBS Calculator v2.0’s biophysical model of translation initiation to rationally design synthetic ribosome binding site sequences39.
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