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
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- 作者：Dr. Huan Xu ; Dr. Yan Wang ; Dr. Jiaqi Lu ; Dr. Bo Zhang ; Ziwei Zhang ; Dr. Longlong Si ; Ling Wu ; Tianzhuo Yao ; Dr. Chuanling Zhang ; Prof. Sulong Xiao ; Prof. Lihe Zhang ; Prof. Qing Xia and Prof. Demin Zhou
- 刊名：ChemBioChem
- 出版年：2016
- 出版时间：July 1, 2016
- 年：2016
- 卷：17
- 期：13
- 页码：1250-1256
- 全文大小：1190K
- ISSN：1439-7633
文摘
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.