title: 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.
authors.affiliation: State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, No. 38 Xueyuan Road, Beijing, 100191, China.
authors.forename: Huan
authors.initials: H
authors.lastname: Xu
authors.affiliation: State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, No. 38 Xueyuan Road, Beijing, 100191, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, No. 38 Xueyuan Road, Beijing, 100191, China.
authors.forename: Jiaqi
authors.initials: J
authors.lastname: Lu
authors.affiliation: State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, No. 38 Xueyuan Road, Beijing, 100191, China.
authors.forename: Bo
authors.initials: B
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, No. 38 Xueyuan Road, Beijing, 100191, China.
authors.forename: Ziwei
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, No. 38 Xueyuan Road, Beijing, 100191, China.
authors.forename: Longlong
authors.initials: L
authors.lastname: Si
authors.affiliation: State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, No. 38 Xueyuan Road, Beijing, 100191, China.
authors.forename: Ling
authors.initials: L
authors.lastname: Wu
authors.affiliation: State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, No. 38 Xueyuan Road, Beijing, 100191, China.
authors.forename: Tianzhuo
authors.initials: T
authors.lastname: Yao
authors.affiliation: State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, No. 38 Xueyuan Road, Beijing, 100191, China.
authors.forename: Chuanling
authors.initials: C
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, No. 38 Xueyuan Road, Beijing, 100191, China.
authors.forename: Sulong
authors.initials: S
authors.lastname: Xiao
authors.affiliation: State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, No. 38 Xueyuan Road, Beijing, 100191, China.
authors.forename: Lihe
authors.initials: L
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, No. 38 Xueyuan Road, Beijing, 100191, China. xqing@bjmu.edu.cn.
authors.forename: Qing
authors.initials: Q
authors.lastname: Xia
authors.affiliation: State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, No. 38 Xueyuan Road, Beijing, 100191, China. deminzhou@bjmu.edu.cn.
authors.forename: Demin
authors.initials: D
authors.lastname: Zhou
mesh_terms: D000596:Amino Acids / Q000235:genetics*; D001386:Azides; D003062:Codon; D003978:Diazomethane / Q000031:analogs & derivatives; D004926:Escherichia coli / Q000235:genetics*; D005697:Galactosides / Q000737:chemistry; D005818:Genetic Engineering; D007211:Indoles / Q000737:chemistry; D007763:Lac Operon; D008239:Lysine / Q000031:analogs & derivatives / Q000235:genetics; D001616:beta-Galactosidase / Q000737:chemistry
chemical_list: C000622619:((3-(3-methyl-3H-diazirine-3-yl)propamino)carbonyl)-Nepsilon-lysine; D000596:Amino Acids; D001386:Azides; D003062:Codon; D005697:Galactosides; D007211:Indoles; C000622618:N-epsilon-2-azidoethyloxycarbonyllysine; D003978:Diazomethane; D001616:beta-Galactosidase; D008239:Lysine; C044888:5-bromo-4-chloro-3-indolyl beta-galactoside
keywords: amber codon; blue-white screening; codon context; genetic code expansion; high-throughput screening; protein engineering