Re‐exploration of the Codon Context Effect on Amber Codon‐Guided Incorporation of Noncanonical Amino Acids in Escherichia coli by the Blue–White Screening Assay

Abstract

The effect of codon context on amber codon-guided incorporation of noncanonical amino acids (NAAs) has been previously examined by antibiotic selection. Here, we re-explored this effect by screening a library in which three nucleotides upstream and downstream of the amber codon were randomised, and inserted within the lacZ-α gene. Thousands of clones were obtained and distinguished by the depth of blue colour upon exposure to X-gal. Large-scale sequencing revealed remarkable preferences in nucleotides downstream of the amber codon, and moderate preferences for upstream nucleotides. Nucleotide preference was quantified by a dual-luciferase assay, which verified that the optimum context for NAA incorporation, AATTAGACT, was applicable to different proteins. Our work provides a general guide for engineering amber codons into genes of interest in bacteria.

Two‐Tier Screening Platform for Directed Evolution of Aminoacyl–tRNA Synthetases with Enhanced Stop Codon Suppression Efficiency

“…A dual vector system, in which one plasmid directs the expression of the reporter protein (β-galactosidase or YFP) and a second plasmid encodes for the AARS/tRNA pair, facilitates the transfer of the AARS-encoding plasmid from the Tier-2 screen to the YFP-based functional assay. Interestingly, although the LacZ gene was previously used as a reporter for amber stop codon suppression, [20] it has never been applied to guide the engineering of AARS enzymes.…”

Rationally designed Spot 42 RNAs with an inhibition/toxicity profile advantageous for engineering E. coli

“…The lacZ ‐α peptide gene (BBa_I732020) was coupled to constitutive promoter BBa_J23110 in medium copy pSB3K3 (kanamycin R). Based on the rationale of Xu et al, a UAG codon was inserted in frame between codons 6 and 7 by inverse PCR mutagenesis . In cells transformed with two different plasmids, agarose gel electrophoresis was used to confirm that the relative copy numbers of the plasmids were roughly as expected.…”

Dissecting the Contribution of Release Factor Interactions to Amber Stop Codon Reassignment Efficiencies of the Methanocaldococcus jannaschii Orthogonal Pair

“…Three recent works have presented data that can be analyzed to shed light on the context-specific reading of amber codons by the M. jannaschii and Methanosarcina mazei orthogonal tRNA/aaRS systems most commonly used in genetic code expansion [ 12 , 38 , 39 ]. Two of these studies were directed at generating improved systems for ncAA incorporation; however, the data presented can also be used to evaluate the effect of codon context on the efficiency of translation.…”

“…Codon context effects were explicitly examined for the M. mazei pyrrolysyl tRNA/aaRS orthogonal pair by Xu et al employing a β-galactosidase assay to evaluate suppression efficiencies of a library containing randomized codons upstream and downstream of the targeted UAG codon (NNN-UAG-NNN) [ 38 ]. The overall consensus sequence for strong amber stop codon suppression identified was contained within the consensus sequence selected by the Pott et al evaluation.…”

“…The overall consensus sequence for strong amber stop codon suppression identified was contained within the consensus sequence selected by the Pott et al evaluation. The Pott et al evaluation identified additional, stronger sequence effects based on the identity of the nucleotides at positions −6, −5, +7, +8 and +9, sites not analyzed by Xu et al In the Xu et al study [ 38 ], the efficiency of suppression across the evaluated sequence contexts varied over a 100-fold range, much larger than had been previously observed for natural suppressors. The relative importance of specific bases was difficult to interpret as many of the single base mutants were not reported.…”