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Tight translational control using site-specific unnatural amino acid incorporation with positive feedback gene circuits Yusuke Kato ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.8b00204 • Publication Date (Web): 06 Jul 2018 Downloaded from http://pubs.acs.org on July 11, 2018

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ACS Synthetic Biology

Tight translational control using site-specific unnatural amino acid incorporation with positive feedback gene circuits Yusuke Kato*

ABSTRACT

Tight regulatory system for gene expression, which is ideally controlled by unnatural and bio-orthogonal substances, is a keystone for successful construction of synthetic gene circuits. Here, we present a widely applicable approach to construct tight protein translational switches using site-specific unnatural amino acid (Uaa) incorporation systems. As a key mechanism to obtain excellent tightness, we installed gene circuits for positive feedback derepression. This mechanism dramatically suppressed leakage translation in the absence of the Uaa. In a translational switch with the feedback circuit in Escherichia coli, a 1.4 × 103 ON/OFF ratio was achieved which was 3 × 102-fold greater than that of the parent system and was comparable to that of the well-known tight expression system using the araBAD promoter and the araC regulator. This method offers an avenue for generation of novel tight genetic switches from over a hundred site-specific unnatural amino acid incorporation systems which have already been established. These tight translational switches will facilitate the development of fine gene control systems in synthetic biology, especially for Uaa-auxotrophy-based biological containments and live attenuated vaccines.

Keywords: Unnatural amino acids, Translation, Escherichia coli, Genetic switch, Positive feedback derepression, Stop codon readthrough

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INTRODUCTION

Genetic parts do not often operate in an ideal manner whereas most electronic parts work precisely1. It is still challenging to design synthetic gene circuits that function in a predictable manner2. Conditional and gene-specific expression control is a key technology for constructing such gene circuits, and the performance of the expression control system influences that of the entire circuit. One of the most important requirements for expression control is “tightness” which means full expression under the induction condition and near-zero leakage expression under the non-induction condition3. Such tightly controlled inducible expression systems are continuously demanded to develop. Site-specific incorporation of an unnatural amino acid (Uaa) is a technology that allows incorporation of Uaa’s into ribosomally synthesized proteins at an unassigned codon, typically the UAG amber stop codon4-10. This system consists of an aminoacyl-tRNA synthetase (aaRS) which is modified to specifically recognize an Uaa as a substrate (UaaRS) and its cognate amber suppressor tRNA (tRNACUA). Host cells can acquire the ability to incorporate an Uaa by introduction of the UaaRS/tRNACUA genes. The UaaRS/tRNACUA pairs are generated from aaRS/tRNA pairs, which are usually derived from evolutionarily distant organisms and are orthogonal to those of the host, i.e., the UaaRS does not catalyze any host tRNAs and its cognate tRNA does not charge any amino acids by the host enzymes. The Uaa is exclusively incorporated at UAGs in the host cells which maintain the UaaRS/tRNACUA pairs if the orthogonality is perfect. Site-specific Uaa incorporation was originally developed to modify target proteins, such as for structural analysis, labeling, and functional modification of proteins4-6. This system has also been used as a genetic switch to control target gene expression at the translational level11,12. One or more TAGs (UAGs in mRNAs) are inserted in the open reading frame of the target genes to construct this translational switch. Extracellularly supplied Uaa’s are transported into the intracellular space and then turn on the translational switch. In the ON-state, the UAGs are subsequently suppressed by the Uaa incorporation causing stop codon read through, resulting in the entire translation of the target transcripts. In the OFF-state where the Uaa is not present, the translation terminates at the inserted UAGs. The expression of target genes can be controlled using the translational switch if the truncated products lose their functions. UAG insertions next to the translation start codon can ensure the loss-of-function and prevent the accumulation of truncated products. The translational switch has been noteworthy for its use in biological containment systems employing synthetic auxotrophy for an Uaa. Biological containment is a genetic program for restricting organism growth only in human-controlled areas, such as the laboratory


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and factory, and results in the death of the organism in the natural environment13. This technology is necessary for safety control for “useful, but dangerous” organisms, such as genetically modified organisms whose safety has not been certified, pathogens, and harmful invasive species13,14. Recently, some papers have reported the bacterium Escherichia coli which is engineered to survive only in the presence of an Uaa, as a novel biological containment system13,15-17. In this bacterium, the essential genes for survival, which are either naturally existing genes in the genome or synthetic essential genes such as anti-toxin genes against co-introduced toxin genes, need the Uaa for expression. The translational switch is used as an essential genetic part in this system. A similar technology has also been used for generation of the safe and effective live-virus vaccines, such as vaccines for a human immunodeficiency virus and an influenza virus18-20. These attenuated viruses can proliferate only in the cells which are engineered to maintain the Uaa incorporation system and in the presence of the Uaa whereas they cannot replicate in wild-type cells. The site-specific Uaa incorporation system was not originally developed as a translational switch, and the tightness is not assured. In addition, even if the UaaRS/tRNACUA pair is highly specific for its substrate Uaa, the tightness will be affected by various factors, such as the expression strain, their level of expression and the sequence context of UAG6,21. The tightness is indeed varied among the translational switches as shown below, suggesting that methods to build a tight switching device using the Uaa incorporation system are needed. Here, we present a highly adaptable approach for constructing tight protein translational switches using site-specific Uaa incorporation systems. These tight translational switches will facilitate the development of the biological containments using Uaa-auxotrophy, live attenuated vaccines, and fine gene control systems in synthetic biology.

RESULTS AND DISCUSSION Two components of leakage translation Our goal is to establish a general method to construct tight translational switches with both a leakage translation of almost zero in the OFF-state and maximum translation in the ON-state.

We developed a method using an incorporation system involving the Uaa

3-iodo-L-tyrosine (IY), which was derived from the tyrosyl-RS/tRNA pair of the archaeon Methanocaldococus jannaschii, as a model22. This translational switch consists of the IY-specific aaRS (IYRS) and its cognate tRNACUA (MJR1). The plasmid containing these genes was transfected into Escherichia coli BL21-AI (Figure S1). We also constructed a reporter gene which allows evaluation of the translational efficiency over a wide range (Figure S2). This reporter gene encodes EGFP which is tagged with a V5-epitope at the N-terminus. A single TAG



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was inserted next to the translation initiation codon in the V5-epitope region (1a-V5-EGFP). The product of the reporter gene can be easily quantified by EGFP fluorescence. In addition, quantitative western blot analysis can also be used to detect low levels of leakage expression in the OFF-state. This reporter gene is contained within another plasmid. The properties of translational switches were studied using an E. coli strain maintaining both plasmids that encode the switch components and the reporter gene. We first evaluated leakage translation of the reporter protein in the absence of IY by western blot analysis (Figure 1A). Although substitution of the translational switch encoded plasmid with an empty vector reduced the leakage, a detectable level of reporter protein was still observed, suggesting that the leakage translation consisted of two components.

One

component is the leakage translation attributable to the switch (LTaS). Incomplete orthogonality of IYRS could cause a natural amino acid mischarge to MJR1, resulting in LTaS. Another component is the leakage translation attributable to the basal read-through (LTaBR) which is independent from the function of the translational switch. This component is mainly caused by basal read-through by near cognate tRNAs. The UGA stop codon of the Qβ protein mRNA was read-through at a level of 3-4%23. A similar basal read-through has also been reported for UAG24. Both the LTaS and LTaBR, therefore, must be suppressed to achieve a tight translational switch. Suppression of the LTaS by positive feedback derepression We constructed a positive feedback derepression circuit for IYRS gene expression in the translational switch to suppress LTaS (Figure 1B). This circuit derepressed IYRS gene expression dependent on IY-MJR1 which was the output of the translational switch. The expression of IYRS would decrease in the absence of IY, i.e., the OFF-state, resulting in reduced generation of mischarged MJR1, which resulted from IYRS catalysis (Figures 1C and S3). Finally, LTaS would be suppressed because the mischarged MJR1 mainly causes the LTaS. In contrast, the translation of IYRS would be derepressed in the presence of IY, i.e., the ON-state, and the target gene transcripts would be maximally translated. The easiest method to construct the feedback circuit is the insertion of one or more TAGs in the coding region of the IYRS gene (Figure 1D). We first constructed an IYRS derivative gene, 1a-IYRS, in which a single TAG was inserted as the simplest feedback circuit (Figures 2A and S2). This TAG-inserted IYRS expression construct would not generate a truncated and premature IYRS because the TAG was located next to the translation initiation site, ATG. The LTaS and the maximum expression (ME), which was induced the optimal concentration of IY, were evaluated (Figures 2B,2C and S4). The LTaS for 1a-IYRS decreased to 30% of that of the parent IYRS. On the other hand, the ME also decreased to 51%. We evaluated the translational switch tightness using “net gain” which was defined as the ME/LTaS. Disappointingly, the net


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gain for 1a-IYRS was 8.2 which was only a 1.7-fold increase over that of its parent IYRS (Figure 2D). However, an additional modification involving a Met 6 Val substitution [1a-IYRS(M6V)] more significantly suppressed the LTaS, and the net gain was improved to a 14-fold increase (Figure 2A). The Met 6 may be an alternative translation initiation site and may cause leaky expression of IYRS even in the OFF-state. The LTaS, ME, and net gain of the double and triple TAG insertion derivatives [2a- and 3a-IYRS(M6V)] were similar to those of 1a-IYRS(M6V). Quadruple TAG insertion [4a-IYRS(M6V)] yielded the strongest suppression of the LTaS and the best net gain, 27-fold over that of the parent IYRS, whereas the ME decreased to 1/3 of that of 1a-IYRS(M6V). Although the quadruple TAG insertion in the parent IYRS (4a-IYRS) also suppressed the LTaS, the net gain was less than that of 1a- to 3a-IYRS(M6V). Another method to construct a feedback circuit is via transcriptional control using cis-regulatory leader-peptide elements which are controlled by an Uaa25 (Figure 1E). This regulatory element, UAAon, which is inserted into the IYRS gene is expected to repress the transcription of this gene in the OFF-state. The LTaS was significantly suppressed by insertion of UAAon into the IYRS gene (Uo-IYRS) whereas the ME more clearly decreased, and the net gain was less than that of the Met 6 Val derivatives (Figure 2B,2C and 2D). Feedback to the MJR1 gene is also predicted to suppress transcription. Only the feedback using UAAon can be used for the MJR1 gene because the functional product of this gene is RNA. UAAon insertion in the MJR1 gene (Uo-MJR1) exhibited a result similar to that of Uo-IYRS (Figure 2B,2C and 2D). A double feedback to both IYRS and MJR1 [2a-IYRS(M6V) + Uo-MJR1] was also tested. No significant LTaS was detected although the ME decreased to 37% of that of 2a-IYRS(M6V) (Figure 2B and 2C). Although 4a-IYRS(M6V) and 2a-IYRS(M6V)+UoMJR1 exhibited higher values of net gain, since 2a-IYRS(M6V) was one of the best translational switches which successfully both maintained the ME and achieved a good net gain, further development was made using 2a-IYRS(M6V). Suppression of the LTaBR by multiplexed TAG insertion The LTaBR is another component of leakage translation, indicating that the LTaBR also needs to be suppressed to obtain the least amount of leakage translation. An increase in the number of TAG insertions in the target genes more effectively suppresses the read-through mechanisms which are less competitive against the peptide release factor RF1, suggesting that the LTaBR, which is mainly attributed to natural amino acid incorporation by the near cognate tRNAs, will be more strongly reduced than the ME11,26. The LTaBR with both two and three TAG-inserted reporter genes (2a- and 3a-V5-EGFP) was evaluated in the strains which did not contain an Uaa incorporation system (Figures 3A and S2). The dramatic decrease in the level of the LTaBR correlated with the number of inserted TAGs, as expected.



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Next, we tested the effect of multiplexed TAG insersion in 2a-IYRS(M6V) strains. The ME of 2a-V5-EGFP remained at 44% of that of 1a-V5-EGFP whereas that of 3a-V5-EGFP sharply decreased to 2.4% (Figure 3B). Both 2a-IYRS(M6V) and 2a-V5-EGFP were selected as the best possible combination. For practical use, the performance of translational switches should be evaluated using “gross gain” which is defined as the ME/(LTaS + LTaBR). The gross gain of the combination of 2a-IYRS(M6V) and 2a-V5-EGFP, which was evaluated by quantitative 3

western blot, was 1.4 × 10 which was more than a 10-fold increase over that of 1a-V5-EGFP -2

(Figure 3C). The ME of this combination was estimated as 3 × 10 g/L after a 6 h incubation in a shaker flask (Figure S5). Tunability, response rate, and reversibility A dose-response curve was evaluated for 2a-IYRS(M6V) to test its tunability (Figure -4

4A). The production of EGFP reached maximum levels with 3 × 10 M IY. Intermediate levels of EGFP production were observed with suboptimal concentrations, approximately spanning a -6

-4

2-log10 concentration range from 3 × 10 to 3 × 10 M. Such intermediate-level expression was also observed in the parent IYRS and confirmed in individual bacteria, suggesting that the translational switch using 2a-IYRS(M6V) is tunable, i.e., possible to control the translational efficiency at any intermediate magnitude by adjustment of the IY concentration12. This result also suggests that dose-response curves may be determined prior to use to obtain the best performance of the Uaa-translational switches as intended. Next, the response rate of OFF/ON transition was assessed (Figure 4B). EGFP fluorescence increased within 10 min after addition of the optimal concentration of IY and reached a maximum production rate within 40-60 min. This result was similar to that of the parent IYRS, suggesting that the modification in 2a-IYRS(M6V) was less affective on the response rate11. Reversibility was also tested (Figure 4C). Washout of extracellularly supplied IY decreased EGFP fluorescence within 30-60 min presumably mainly by dilution-by-proliferation, suggesting that this translational switch can transit from the On-state to the OFF-state. The lag time could be due to the accumulation of IY in bacterial cells13. Adding back IY induced an increase in the EGFP fluorescence again, suggesting that this translational switch is completely reversible. Effect of positive feedback derepression circuits in another UaaRS/tRNACUA system Pyrrolysine is a lysine derivative encoded by UAG in certain methanogens and is often referred to as the 22nd genetically encoded natural amino acid27. Pyrrolysyl-tRNA synthetase (PylS)/ its cognate tRNACUA (PylT) pair and their derivatives have also been used for site-specific Uaa incorporation in E.coli6,28. The Uaa Nε-benzyloxycarbonyl-L-lysine (ZK) incorporation system was developed from PylS/PylT of the archaeon Methanosarcina mazei29. The


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translational switch using ZK incorporation could control the translational efficiency in either an all-or-none manner or at any intermediate magnitude in individual bacteria, as confirmed using IY incorporation12 (Figures S6 and S7). The effect of a positive feedback derepression circuit in this translational switch was assessed. It is noteworthy that the LTaS was low, only 2% of the ME, even for the parent ZK-specific aaRS (ZKRS)/PylT pair (Figures 5A and S2). The LTaS was significantly suppressed by addition of the feedback circuit to both the ZKRS and pylT genes using UAAon whereas the ME also decreased to 1/3-fold and 1/2-fold of that for the parent, respectively (Figures 5A and B). Although net gains could not be estimated because a significant level of the LTaS was not detected with the strains involving the feedback circuits, the suppression level of the LTaS was apparently greater than that of the ME, suggesting that the positive feedback circuits also improved the tightness of the translational switches using the PylS/PylT derivatives.

CONCLUDING REMARKS Table 1 contains a summary of the properties of selected translational switches which were evaluated in this study. The best translational switch, involving the combination of 3

2

2a-IYRS(M6V) and 2a-V5-EGFP, achieved a gross gain of 1.4 × 10 which was 3 × 10 -fold greater than that of the parent system. This gross gain is comparable to that of the araBAD promoter/araC repressor system which is probably the best-known tight expression system in E. coli30. Althogh the performances of Uaa translational switches are difficult to directly compare to each other because various factors affect it as mentioned above, the gross gains of some well-characterized Uaa incorporation systems were calculated to be several to several ten1,11,12,21,31,32. These results suggest that the positive feedback gene circuit is a highly efficient method to optimize a site-specific unnatural amino acid incorporation system for use as a translational switch. This translational switch not only allows for all-or-none switching but also can control the translational efficiency at any intermediate magnitude by adjustment of the IY concentration in the medium. An OFF/ON transition was complete within ten minutes, and removal of IY evoked an ON/OFF transition. These results indicate that these translational switches are tight, tunable, able to respond rapidly, and reversible. In principle, the positive feedback derepression circuit to generate tight translational switches is applicable for any UaaRS/tRNACUA pairs. For example, this method was effective in two major orthogonal UaaRS/tRNACUA pairs, M. janaschii TyrRS/MJR1 and M. mazei PylS/PylT, which are widely used in E. coli. To date, over 150 non-canonical amino acids, including Uaa’s, have been ribosomally incorporated into E. coli proteins33. Similar site-specific Uaa incorporation systems have been used not only for E. coli, but also for other bacteria, yeasts, nematodes,



insects, mammals, and plants (exhaustively summarized in ref.7). This method offers an avenue for generation of novel tight genetic switches from over a hundred site-specific unnatural amino acid incorporation systems which have already been established.

MATERIALS AND METHODS Strains, growth conditions, and transfection E. coli BL21-AI[F ompT gal dcm lon hsdSB(rB mB) araB::T7RNAP-tetA] was used throughout this study34. E. coli XL1-Blue and E. coli DH5 were also used for plasmid construction. The bacteria were grown in Luria-Bertani (LB) medium at 37°C and 200 r.p.m for all experiments. Carbenicillin (100 µg/ml), chloramphenicol (50 µg/ml), and kanamycin (50 µg/ml) were added as appropriate. Transfection was performed by electroporation using a Gene Pulser IITM electroporator (BIO-RAD). Plasmid construction The plasmids are listed in Figure S2.

The plasmid pTYR MjIYRS2-1(D286) MJR1 × 3

encoding parent IYRS and MJR1, and pTK2-1 ZLysRS1 encoding parent ZKRS and PylT were provided from Kensaku Sakamoto and Shigeyuki Yokoyama (RIKEN)22,29. A single or multiplexed TAG insertion next to the translation initiation site of IYRS for the construction of positive feedback derepression circuits was prepared using inverse PCR with TAG-added and 15-20 bp overlapped primers, followed by circularization using enzymatic recombination (In-Fusion HD cloning kit, Takara). PCR was performed using a high fidelity DNA polymerase (KOD-plus, TOYOBO) for all experiments. IYRS(M6V) was generated by a similar method using inverse PCR. The UAAon transcription control element was custom synthesized by GenScript. To construct Uo-IYRS, an inverse PCR product, which was cleaved between the promoter and the IYRS coding region, was fused to a PCR-amplified UAAon element. Other UAAon-inserted UaaRS and tRNACUA genes were also prepared using similar methods. The construction of the 1a-V5-EGFP reporter gene was previously reported12. The inverse PCR and enzymatic circularization mentioned above were used to generate 2a- and 3a-V5-EGFP. Unnatural amino acids (Uaa’s) The Uaa’s were purchased from the following sources: IY, Watanabe Chemical Industries; ZK, Bachem. IY was directly dissolved in LB medium for experimental use. ZK was first dissolved in 1 N HCl at 100 mM and then diluted in LB medium at the final concentration of 3 mM or less. The working ZK solution was neutralized using 1 N NaOH. EGFP reporter assay For fluorescence measurement of pooled bacteria, an overnight (approximately 16 h) culture of bacteria was resuspended in an equal volume of liquid LB medium with or without an Uaa. After a 6 h culture (2 ml in a 14 ml-culture tube), the bacteria were collected by


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centrifugation (10,000 × g for 1 min). The pellet was washed and was resuspended in an equal volume of 0.9% (wt/vol) NaCl, and this wash step was repeated twice. An aliquot of the bacterial suspension (200 µl) was diluted in 3.0 ml of 0.9% NaCl, and the OD590 was measured. The fluorescence intensity of the bacterial population was measured using a Shimadzu RF-5300PC spectrofluorometer (excitation, 480 nm; emission, 515 nm). The background fluorescence was estimated using bacteria containing an empty vector (pACYC184). For the time course measurement of EGFP accumulation, the bacteria were cultured in 20 ml LB medium in a 250-ml culture flask. Aliquots of the bacterial culture (200 µl) were withdrawn at various time points, and the OD590 and fluorescence were measured. The methods for preparing the photomicrographs and image analyses were previously reported in detail12. Photomicrographs and Nomarski differential interference contrast images of the fluorescent bacteria were recorded using both a Carl Zeiss Axioskop 2 with a 38-HE Endow GFP flter-set (Carl Zeiss) and a Roper Scientifc Photometrics CoolSNAP ver.1.1 (Roper Industries). Analyses of the fluorescent images were performed using ImageJ 1.48v (National Institutes of Health). Prior to analyses, the fluorescent images were confirmed as not being saturated at any pixels. Three-dimensional graphs of the intensities of the pixels were generated using Surface Plot command. The fluorescent intensity of individual bacteria was measured using Particle Analysis command. A background value (bacteria-absent area) was used as a threshold for particle detection. A range of particle areas was set to detect only individual and not-overlapping bacterial cell images. Western blot Bacteria were collected from a 1.5 ml bacterial suspension by centrifugation (10,000 × g for 1 min). The pellet was washed and was resuspended in an equal volume of 0.9% (wt/vol) NaCl, and this wash step was repeated three times. Finally, the bacteria were resuspended in 150 µl of 0.9% NaCl. An aliquot of the bacterial suspension (20 µl) was analyzed by measurement of the OD590. Another aliquot (120 µl) was mixed with 2x Laemmli’s sample buffer containing β-mercaptoethanol and then sonicated. The bacterial lysates were boiled for 5 min at 100 °C. The samples were separated by SDS-page using a 10-20% acrylamide gradient gel (ATTO). The separated proteins were transferred to a PVDF membrane using a semi-dry blotter (Trans-Blot SD semi-dry transfer cell, BIO-RAD). Western blot was performed with an alkaline phosphatase conjugated anti-V5 monoclonal antibody (Anti-V5-AP antibody, 46-0287, Thermo Fisher Scientific) and a chemiluminescence detection (SignaLOCK ChemiWestern kit, AP, for Film/Imager, SeraCare) according to the manufacturer’s protocol. Proteins were visualized and quantified using an image analyzer (LAS-3000 mini, Fujifilm).

A constitutively detected band

around 27 kDa, which could be detected by Commasie blue staining, was used as a loading control35.



To determine the gross gain, a three-fold dilution series of the ON-state and the OFF-state samples was evaluated. Data were normalized to the loading control protein band. A standard curve was generated by plotting the data for the ON-state samples. The relative expression level at the OFF-state was determined, and then the gross gain was calculated. The yield of V5-EGFP protein was estimated using a control V5-tagged protein (PositopeTM, Thermo Fisher Scientific). Similar to the procedure for the determination of gross gain, a three-fold dilution series of samples and Positope were evaluated. A standard curve from the data for Positope was generated, and then the protein yield per liter culture broth was calculated (see also Figure S5). Statistics Statistical analyses were performed using Welch’s t-test in Excel ver.14.0.

ASSOCIATED CONTENT Supporting Information Figure S1 – S7

ABBREVIATIONS Uaa, unnatural amino acid; aaRS, aminoacyl-tRNA synthetase; UaaRS, aminoacyl-tRNA synthetase (aaRS) which is modified to specifically recognize an Uaa as a substrate; IY, 3-iodo-L-tyrosine; LTaS, the leakage translation attributable to the switch; LTaBR, the leakage translation attributable to the basal read-through; ME, maximum epression; ZK, Nε -benzyloxycarbonyl-L-lysine.

AUTHOR INFORMATION Division of Biotechnology, Institute of Agrobiological Sciences, National Agriculture and Food Research Organization (NARO), Oowashi 1-2, Tsukuba, Ibaraki 305-8634, Japan *E-mail: [email protected]

ACKNOWLEDGMENTS This work was supported by JSPS KAKENHI grant numbers JP25660281 and JP16K08121. We thank Kensaku Sakamoto and Shigeyuki Yokoyama for the plasmids pTYR MjIYRS2-1(D286) MJR1 × 3 and pTK2-1 ZLysRS1.

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Figure 1. Positive feedback derepression circuit. (A) Two components of leakage translation. The parent IYRS/MJR1 pair was tested as a translational switch. The leakage expression of V5-EGFP protein was detected by western blot. The loading control band was detected by Commasie blue staining. (B) Schematic of positive feedback derepression circuit. The feedback circuit is represented in red. (C) Expected behaviors of the feedback circuit. The expected behaviors with/without the positive feedback circuit and in the presence/absence of an Uaa are shown. (D) Feedback by translation control. The feedback to UaaRS gene expression is represented. The expected behaviors in the presence/absence of an Uaa are shown. A single



TAG (shown in magenta) is inserted next to the translational initiation site, ATG. (E) Feedback by transcription control. UAAon (shown in pink) represents a cis-regulatory leader-peptide element which is controlled by an Uaa.


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Figure 2. Suppression of the LTaS by positive feedback derepression. (A) Sequence of 1a-IYRS(M6V). The nucleotide and protein sequences for the N-terminal region are shown. The inserted TAG and the substituted nucleotide for Met 6 Val are highlighted in black and a blue background, respectively. The translational initiation ATG is represented in magenta. (B) LTaS and (C) ME. The accumulation of a target gene (V5-EGFP) was quantified by EGFP fluorescence. The measured fluorescence data were used after subtraction of the negative control (the strain carrying the blank plasmid pACYC184 instead of an Uaa incorporation system) values. All data points represent the mean ± s.e.m. of six biological replicates. Significant differences between the sample fluorescence data and the background fluorescence are indicated for the LTaSs. ns, not significant (P > 0.05). *P < 0.01, **P < 0.001, ***P < 0.0001, ****P



< 0.00001, *****P < 0.000001. (D) Net gain. The net gain was calculated from the mean values of the LTaS and ME shown in (B) and (C). nc, not calculated due to insignificant LTaS.


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Figure 3. Suppression of the LTaBR by multiplexed TAG insertion. (A) Effect of multiplexed TAG insertion. The LTaBRs of a target gene (V5-EGFP), in which 1 to 3 TAGs were inserted next to the translational initiation site, ATG, were detected by western blot. Strains carrying the blank plasmid pACYC184 instead of an Uaa incorporation system were tested. The loading control band was detected by Commasie blue staining. (B) ME. The 2a-IYRS(M6V) strains were tested. The accumulation of a target gene (V5-EGFP) was quantified by EGFP fluorescence. The measured fluorescence data were used after subtraction of the negative control values. All data points represent the mean ± s.e.m. of six biological replicates. (C) Gross gain. The accumulation of V5-EGFP protein in the presence/absence of IY was quantified by western blot, and the gross gain was calculated. All data points represent the mean ± s.e.m. of five biological replicates.



Figure 4. Tunability, response rate, and reversibility. (A) Tunability. An IY dose-response curve for the 2a-IYRS(M6V) + 2a-V5-EGFP strain was evaluated. The concentration range, in which the translational efficiency was intermediate, is shown in pink. The measured fluorescence data were used after subtraction of the negative control values. All data points represent the mean ± s.e.m. of three biological replicates. (B) Response rate. The IY concentration was changed from 0 to 3 × 10-4 M at time 0. The data points represent an increase in fluorescence from time 0. The red line indicates a linear fit of the data points between 60 to 130 min. (C) Reversibility. The ON/OFF and OFF/ON transitions were tested. The presence or absence of IY is indicated in the identical color to that of the symbols and lines. After a 120-min incubation (at time 0 in the graph)


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in the presence of IY, the medium was replaced with fresh medium either containing (open circles) or lacking 3 × 10

-4

M IY (filled circles). The medium was replaced again with an

IY-containing medium after 150 min (filled circles).

Figure 5. Effect of positive feedback derepression circuits in a PylS/PylT-derived translational switch. The translational switches using a ZK incorporation system with/without the feedback circuits were tested. 1a-V5-EGFP was used as a reporter gene. (A) LTaS and (B) ME. The accumulation of a target gene (V5-EGFP) was quantitated by EGFP fluorescence. The measured fluorescence data were used after subtraction of the negative control values. All data points represent the mean ± s.e.m. of six biological replicates. Significant differences between the sample fluorescence data and the background fluorescence are indicated for the LTaSs. ns, not significant (P > 0.05). *P < 0.01.


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