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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. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Citation

Huan Xu, Yan Wang, Jiaqi Lu, Bo Zhang, Ziwei Zhang, Longlong Si, Ling Wu, Tianzhuo Yao, Chuanling Zhang, Sulong Xiao, Lihe Zhang, Qing Xia, Demin Zhou. 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. Chembiochem : a European journal of chemical biology. 2016 Jul 1;17(13):1250-6


PMID: 27028123

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