Tunable translational control using site-specific unnatural amino acid incorporation in Escherichia coli
- Published
- Accepted
- Subject Areas
- Bioengineering, Biotechnology, Microbiology
- Keywords
- Translational regulation, Escherichia coli, Expression system
- Copyright
- © 2015 Kato
- Licence
- This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ PrePrints) and either DOI or URL of the article must be cited.
- Cite this article
- 2015. Tunable translational control using site-specific unnatural amino acid incorporation in Escherichia coli. PeerJ PrePrints 3:e855v1 https://doi.org/10.7287/peerj.preprints.855v1
Abstract
Translation of target gene transcripts in Escherichia coli harboring UAG amber stop codons can be switched on by the amber-codon-specific incorporation of an exogenously supplied unnatural amino acid, 3-iodo-L-tyrosine. Here, we report that this translational switch can control the translational efficiency at any intermediate magnitude by adjustment of the 3-iodo-L-tyrosine concentration in the medium, as a tunable translational controller. The translational efficiency of a target gene reached maximum levels with 10-5 M 3-iodo-L-tyrosine, and intermediate levels were observed with suboptimal concentrations (approximately spanning a 2-log10 concentration range, 10-7 to 10-5 M). Such intermediate-level expression was also confirmed in individual bacteria.
Author Comment
This is a submission to PeerJ for review.
Supplemental Information
Figure S1. Sequence of an amber-inserted EGFP expression construct
The amber-inserted EGFP expression sequence was cloned into pDONR221 using a standard Gateway reaction by BP recombination.
Fig. S2. EGFP fluorescence of bacteria carrying the ΔMJR1 negative control plasmid
A bacterial strain carrying both the ΔMJR1 and the amber-inserted EGFP expression plasmid was evaluated. EGFP fluorescence was measured at various IY concentrations. Data are shown as mean SEM. n = 3 independent experiments. Statistical analysis was performed using Welch’s t-test (α = 0.05). No significant differences were detected in EGFP fluorescence intensity.
Fig. S3. Growth curves at an extremely high IY concentration
(A) pTYR MjIYRS2-1(D286) MJR13 and the amber-inserted EGFP expression plasmid. (B) pTYR MjIYRS2-1(D286) MJR13 alone. (C) The parental strain BL21-AI without any plasmids. Open circle, 0 M; filled circle, 3 10-3 M.
