Designing Synthetic Flexible Gene Regulation Networks Using RNA

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Letter

Designing synthetic flexible gene regulation networks using RNA devices in cyanobacteria Akiyoshi Higo, Atsuko Isu, Yuki Fukaya, and Toru Hisabori ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.6b00201 • Publication Date (Web): 16 Sep 2016 Downloaded from http://pubs.acs.org on September 20, 2016

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ACS Synthetic Biology

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Designing synthetic flexible gene regulation networks using RNA

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devices in cyanobacteria

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Akiyoshi Higoa;b, Atsuko Isua;b, Yuki Fukayaa;b, and Toru Hisaboria;b #

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a

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Technology, Nagatsuta 4259-R1-8, Midori-ku, Yokohama 226-8503, Japan; bCore Research for

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Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST),

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Tokyo 102-0075, Japan

Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of

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#

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Research, Tokyo Institute of Technology, Nagatsuta 4259-R1-8, Midori-Ku, Yokohama

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226-8503, Japan; Tel. 81-45-924-5234; Fax. 81-45-924-5268, email: [email protected]

Corresponding author: Laboratory for Chemistry and Life Science, Institute of Innovative

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ABSTRACT

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In recent years, studies on the development of gene regulation tools in cyanobacteria have been

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extensively conducted towards efficient production of valuable chemicals. However, there is

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considerable scope for improving the economic feasibility of production. To improve a recently

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reported gene induction system using anhydrotetracycline (aTc)–TetR and an endogenous gene

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repression system using small antisense RNA in the filamentous nitrogen-fixing

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cyanobacterium Anabaena sp. PCC 7120 (Anabaena), we constructed a positive feedback loop,

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in which gfp and a small antisense RNA for tetR are controlled by an aTc-inducible promoter.

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GFP expression in this improved system was higher and longer than the system lacking tetR

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repression. In addition, using TetR aptamer and a riboswitch, we succeeded in achieving a

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superior and longer induction of GFP expression even under high-light conditions. Hence,

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efficient gene induction systems were established in Anabaena by designing a gene regulation

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network using RNA-based tools.

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Key words: cyanobacteria, gene regulation system, small RNA, riboswitch

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INTRODUCTION

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By designing artificial genetic circuits, in which regulatory devices are appropriately combined,

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various types of systems have been created to perform specific tasks in living cells since the

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first construction of the genetic oscillator1 and toggle switch.2 Inspired by electrical engineering

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and computer science, researchers working in the field of synthetic biology have produced

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biological counters,3 Boolean logic gates,4 band-path filters,5 and memory.6 In addition, such

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synthetic biology approach has been applied to the development of biosensors and production of

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valuable chemicals7

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Although cyanobacteria have been expected to play a role in the production of useful

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substances and biofuels because of their photosynthetic ability,8 lack of genetic devices for

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efficient gene regulation in these organisms has limited their productivity.9, 10 To overcome this

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challenge, studies on development of gene regulation tools have been actively conducted in

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recent years.8 Among them, gene induction system consisting of an inducer, anhydrotetracycline

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(aTc), and a transcriptional repressor, TetR, showed a wide dynamic range in some

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cyanobacteria, although photolability of aTc limited the usefulness of this system.11–13 A

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theophylline riboswitch-based gene induction system has also been successfully used in

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cyanobacteria.14, 15 In addition, gene repression systems have been developed in cyanobacteria.

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A small RNA based on an Escherichia coli IS10 RNA-IN/OUT system was used for the

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repression of exogenous gfp expression.12 Furthermore, we and the other group succeeded in

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controlling endogenous gene expression by using artificial small antisense RNA (asRNA)13 and

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CRISPRi system,16 respectively. However, the number of studies on construction of a genetic

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network using such gene regulation devices is limited. Recently, Immethun et al. built a

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two-input AND gate in the unicellular cyanobacterium Synechocystis sp. PCC 6803,17

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demonstrating the potential of synthetic biology in cyanobacteria.

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For the industrial production of valuable materials in cyanobacteria, it is important to

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consider the cost of production. Because synthetic inducers are generally expensive, the amount

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of these inducers used to induce gene expression should be minimal to reduce production costs.

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In addition, photolability-related issues of aTc should be solved when TetR system is used in

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photosynthetic cyanobacteria. Hence, we improved our previous TetR system in Anabaena13

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using a synthetic biology approach. We designed a positive feedback loop and succeeded in

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achieving greater and prolonged gene induction using only a small amount of aTc. Furthermore,

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by combining a TetR aptamer that inactivates TetR repression activity and a riboswitch that

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terminates transcription in the presence of adenine, we enabled strong and prolonged gene

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induction under high-light conditions.

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RESULTS AND DISCUSSION

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In our previous study, we demonstrated that the amount of TetR expressed in cells is a

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determinant of responsiveness to the inducer aTc.13 Thus, tetR was expressed under the control

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of nirA promoter, which is active in the presence of nitrate and is repressed in the absence of a

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nitrogen source.18 Therefore, gene expression is induced under nitrogen-fixing conditions13

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(pCA004 in Figure 1 and supplemental Table S1). In the pCA004 system, induction by nitrogen

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starvation as well as by aTc was possible. Lowering the TetR amount by the promoter

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engineering of PnirA facilitated the creation of a strong induction system (pCA00513), which,

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however, had a narrow dynamic range. In this study, we designed a positive feedback loop to

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induce gene expression strongly with a wide dynamic range. We combined two systems, TetR

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induction and small asRNA repression, which were previously developed by us.13 A small

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asRNA was designed to repress tetR expression, and the small asRNA and the reporter gene gfp

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were expressed under the control of PL03, which is regulated by aTc and TetR (pCA011 in

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Figure 1 and supplemental Table S1). In the case of the pCA011 system, once these genes are

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induced, the amount of TetR in the cell should severely decrease and consequently significant

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gene induction should occur. Here, we expressed TetR with a protease tag LVA13 at the

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C-terminus for the efficient turnover of this protein in the cell (Figure 1).

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We introduced two plasmids, pCA004 that lacked the asRNA gene for tetR repression

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and pCA011, into Anabaena. We then measured GFP fluorescence in these two strains, which

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was induced by indicated concentrations of aTc for 24 h in nitrate-replete (+N) medium (Figure

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2). In addition, we measured GFP fluorescence induced by nitrogen starvation (−N) for 72 h as

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well. GFP fluorescence was very low without the inducer in +N conditions in each strain

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(Figure 2, #1 and #2). As expected, when GFP expression was induced by aTc or nitrogen

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starvation, the asRNA-expressing strain showed stronger fluorescence than the control strain.

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However, even in the presence of 2,000 ng/ml aTc, the pCA011-contaning strain showed only

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one-fourth of GFP fluorescence in a strain without tetR (pCA00113), indicating that a further

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improvement is possible. Western blotting analysis confirmed that the amount of TetR

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decreased in response to aTc in the asRNA-expressing strain but not in the control strain (Figure

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3A). This result demonstrates the plasticity of small asRNA, which can repress a gene in an

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artificial network as well as an endogenous gene.13 When 20 ng/mL aTc was added, the TetR

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amount in the asRNA-expressing strain decreased within 24 h. Even after 72 h, the amount of

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TetR was lower in the asRNA-expressing strain than in the control strain (Figure 3B). In

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addition, GFP expression continued for 72 h in the former strain, while it gradually decreased

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after 24 h in the latter strain (Figure 3B). These results indicate that construction of an artificial

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gene regulatory network complemented photolability of aTc and that such a synthetic biology

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approach is useful in Anabaena.

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Next, we wanted to improve the system in which tetR is expressed under the control of

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PpetE, which would be active irrespective of nitrogen source.19 We designed a similar positive

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feedback loop, in which tetR expression under PpetE is repressed by a small asRNA (pCA012 in

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Figure 1 and supplemental Table S1). A control plasmid pCA002 and pCA012 were introduced

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into Anabaena. We confirmed that the amount of TetR decreased in response to aTc in the

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pCA012-harboring strain (Supplemental Figure S1A). Furthermore, GFP fluorescence in the

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strain was higher than that in the control strain (Supplemental Figure S1B). These results

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suggest that the small asRNA approach has a potential to improve a variety of gene expression

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systems.

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We then constructed a positive feedback loop, in which the amount of TetR is not

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affected but the TetR repressor function is inhibited by TetR-inducing peptide (TiP)20 or TetR

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aptamer.21, 22 A similar design for a prolonged induction had been previously proposed.11 TiP is

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generally used with scaffold proteins such as TrxA from E. coli.20 In this study, we used E. coli

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TrxASS, in which two cysteine residues at the active site were substituted with serine to avoid

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undesirable thiol-disulfide exchange reactions with endogenous proteins in Anabaena.

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Unexpectedly, compared with the control strain, each Anabaena strain containing

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Tip-TrxASS-expressing plasmid pCA013 or TetR aptamer-expressing plasmid pCA014 showed

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no significant differences in GFP fluorescence at various concentrations of aTc (Figure 2, #1, #3,

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and #4). An LVA tag at the C-terminus of TetR or rapid turnover of TetR may interfere with

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stable interaction between TetR and TiP-TrxASS or the TetR aptamer. Next, we expressed the

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TetR aptamer with a tRNA scaffold23 (pCA015 plasmid in Figure 1 and supplemental Table S1)

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to stabilize the TetR aptamer in the cells. While GFP fluorescence in the pCA015-containing

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strain was 5.5-fold higher than that in the control strain under non-induced conditions, the

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former strain exhibited a 9-fold higher GFP fluorescence when induced under nitrate depletion

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than that exhibited by the control strain (Figure 2, #1 and #5). In addition, GFP fluorescence in

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the pCA015-containing strain was higher by induction with, in particular, a small amount of aTc

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(20 ng/mL) than the fluorescence in the control strain. These results indicate that a strong GFP

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induction by aTc or nitrate depletion was achieved, while the pCA015 system was slightly leaky

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compared to the control system.

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Time-course analysis of GFP induction was performed in a tRNA-scaffold TetR

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aptamer-expressing strain using pCA015. In this strain, GFP was strongly expressed even after

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72 h of induction with 20 ng/mL aTc as opposed to the control strain (Figure 4A). When nitrate

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was depleted from the culture medium in both strains, the amount of TetR clearly decreased

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after 8 h (Figure 4B). Unexpectedly, GFP gradually accumulated after 24 h of nitrate depletion

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in these two strains (Figure 4B), in contrast to when GFP was induced by aTc (Figure 4A).

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Development of rapid induction by decrease of TetR amount is required in the future.

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However, such a gradual induction may be preferred in some cases because rapid induction of

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some genes and successful modulation of metabolism immediately after nitrate depletion may

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inhibit the complex heterocyst differentiation process and nitrogen fixation.

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For constructing a flexible gene induction system, we adopted another approach: a

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riboswitch was used to control the expression of tetR. Riboswitch xpt(C74U)/metE,24 an off

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switch that terminates transcription in the presence of adenine, was inserted downstream of PnirA

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in pCA015. The resultant plasmid pCA016 (Figure 1 and supplemental Table S1) was

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introduced into Anabaena. In the pCA16-containing strain, addition of adenine decreased the

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amount of expressed TetR in a concentration-dependent manner (Figure 5A). This result clearly

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indicates that the riboswitch is functional in Anabaena. The amount of TetR after 24 h

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incubation with 250 µM adenine was one-tenth of that in the absence of adenine (Figure 5A).

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Next, GFP was induced by aTc or nitrate depletion both in the presence and absence of adenine.

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After cells were incubated in the presence or absence of 250 µM adenine for 48 h under +N

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conditions, aTc was added to the culture medium and GFP expression was induced for 24 h.

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Furthermore, GFP fluorescence was induced by incubation for 72 h under −N conditions

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irrespective of the presence of adenine. When adenine was not supplied, GFP expression was

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induced by aTc in a concentration-dependent manner (Figure 5B). Adenine alone induced GFP

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fluorescence, and aTc further induced the fluorescence in +N conditions. In addition, adenine

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alone strongly induced GFP under −N conditions. These results indicate that flexible gene

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induction is possible in the pCA016 system by the addition of adequate quantities of two

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inducers, aTc and adenine, and by the selection of specific growth conditions. Supplemental Figure S2 shows microphotographs of the pCA016 strain. Adenine alone

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induced GFP fluorescence sparsely but strongly across the filaments. GFP fluorescence of the

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majority of cells was strongly induced by aTc alone or both of aTc and adenine. These results

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suggest that the pCA016 induction system exhibits all-or-none response rather than a graded

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one.

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When aTc is used as an inducer in gene induction systems, photolability of this molecule

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may be problematic, especially under high-light conditions. Indeed, 20 ng/mL aTc hardly

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induced GFP expression in the pCA015-harboring strain under high-light (150 µmol photons

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m−2 s−1) conditions as opposed to the results under low-light (30 µmol photons m−2 s−1)

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conditions (Figure 6A). Addition of 200 ng/mL aTc induced GFP expression for a short period

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(24 h) in the strain under high-light conditions but at a lesser extent than that induced under

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low-light conditions with addition of 20 ng/mL aTc. To overcome these photolability-related

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problems observed in aTc, we examined the adjusted induction conditions by decreasing the

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amount of TetR via addition of adenine in the pCA016-containing strain. Addition of only 200

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ng/mL aTc hardly induced GFP expression under high-light conditions (Figure 6B, #1). In

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contrast, addition of 250 µM adenine slightly induced GFP after 72 h under high-light

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conditions in this strain (Figure 6B, #2). When both aTc and adenine were added, GFP

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expression was induced after 24 h and its expression continued until 72 h (Figure 6B, #3). When

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adenine was added 24 h before the addition of aTc and irradiation with high light intensities, 20

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or 200 ng/mL aTc induced GFP expression to a greater extent (Figure 6B, #4, and #5) than that

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induced when aTc and adenine were added simultaneously.

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The results shown in Figure 6B indicate that construction of an artificial gene regulatory

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network enabled gene induction with a wide dynamic range in the aTc-TetR system even under

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high-light conditions. The theophylline riboswitch system14, 15 may be alternatively used for

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gene induction under high-light conditions because theophylline is insensitive to light. However,

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the theophylline riboswitch system shows a narrow dynamic range compared with the aTc-TetR

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system (Supplemental Figure S3). In addition, a functional RNA gene could not be induced by

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the theophylline riboswitch system, in which translation is activated in the presence of

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theophylline.14, 15

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In the present study, we demonstrated that purpose-dependent gene regulatory networks

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could be created by simple combination of gene regulation components, TetR and RNA-based

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devices. Complex gene regulation networks consisting of non-coding RNAs and proteins occur

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ubiquitously in nature. For example, similar to the TetR aptamer that sequesters TetR,22 a small

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non-coding RNA, whose function is controlled by a riboswitch, sequesters a regulator protein to

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prevent expression of ethanolamine utilization genes in Firmicutes. 25, 26 Another example was

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recently demonstrated in the unicellular cyanobacterium Synechocystis sp. PCC 6803: a

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feed-forward loop consisting of a transcriptional regulator and a small non-coding RNA that

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prevents translation of target mRNAs is essential for rapid acclimation response to high-light

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stress.27 In addition, development of RNA-based gene regulation tools has been actively pursued

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in cyanobacteria over the past few years12–16, 28, 29 because of their ease of design.30 Inspiration

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from natural systems, as well as electrical engineering and computer science, should make it

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possible to create various types of gene regulation systems, in which genes of interest are

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robustly controlled even under outdoor conditions, by using especially RNA-based devices.

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Construction of such gene regulation networks would help to generate valuable products using

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cyanobacteria in the future.

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METHODS

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Bacterial strains and growth conditions

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Anabaena strains were routinely grown at 30°C at 30–35 µmol photons m−2 s−1 in BG11

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medium31 supplemented with 20 mM HEPES-NaOH (pH 7.5) and 5 µg/mL neomycin sulfate

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unless otherwise stated. For nitrogen starvation experiments, cells were grown in the same

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medium lacking NaNO3 (BG110). Liquid culture was bubbled with air containing 1.0% (v/v)

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CO2.

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Plasmid construction

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DNA fragments were inserted between the EcoRI and BamHI sites of pRL25c,32 a shuttle vector

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replicating in Anabaena (Figure 1). Detailed sequences are described in the Supplemental

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Information.

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Measurement of GFP fluorescence

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GFP fluorescence from Anabaena cultures was measured as described previously.13 Aliquots (3

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ml) of log-phase cultures (OD750 = 0.1–0.2, 48 h after inoculation) of GFP-expressing Anabaena

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strains were moved into 12-well plates with the indicated amount of aTc or with theophylline

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and 10 mM bicarbonate and then incubated for 24 h at 30–35 µmol photons m−2 s−1. Then,

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fluorescence intensities of GFP expressed in Anabaena cultures (OD750 = 0.2–0.4) were

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measured using a fluorescence spectrophotometer FP-8500 (JASCO, Tokyo, Japan) after the

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optical density of Anabaena at 750 nm (OD750) was adjusted to 0.1–0.12 with BG11 or BG110

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medium supplemented with 20 mM HEPES-NaOH (pH 7.5). An excitation wavelength of 488

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nm and an emission wavelength of 510 nm were used. To obtain the expression level in the

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GFP-expressing Anabaena strains, the fluorescence intensity of the cells containing the empty

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vector was subtracted as the background.

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Total protein extraction

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Cultures of Anabaena strains were centrifuged at 4°C at 2,000 g for 3 min. The cell pellets were

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frozen in liquid nitrogen and stored at −80°C until use. The cells were re-suspended in SDS

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sample buffer [125 mM Tris-HCl (pH 6.8), 2% (w/v) SDS, 10% (v/v) glycerol, 6% (v/v)

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2-mercaptoethanol, and 0.01% (w/v) bromophenol blue], and immediately boiled for 5 min.

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After centrifugation at 20,000 g for 10 min, the supernatants were collected as total protein.

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Protein concentration was determined with a CB-X protein assay kit (G-Biosciences, St. Louis,

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MO, USA) using BSA as a standard.

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Western blotting analysis

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Equal amounts of total protein (5 µg and 200 ng for detection of TetR and GFP, respectively)

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obtained from various cell lysates were separated on a denaturing SDS-PAGE gel and blotted

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onto the PVDF membrane. TetR and GFP were detected using a TetR monoclonal antibody

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(Takara Bio, Kusatsu, Japan) and a GFP polyclonal antibody (Sigma-Aldrich, St. Louis, MO,

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USA), respectively, with a chemiluminescent kit (ImmunoStar LD, Wako, Osaka, Japan).

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Fluorescence microscopy

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Fluorescence images were taken on a fluorescence microscopy (model IX73, Olympus, Tokyo,

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Japan) with a mirror unit U-FBNA and U-FGW, for observation of GFP fluorescence and

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phycobiliprotein autofluorescence, respectively. Cultures were bubbled with air containing

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1.0% (v/v) CO2.

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Supporting Information

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A document describing the DNA sequences used in this study, as well as summary of aTc-TetR

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induction systems used in this study in Table S1 and Figure S1-S3.

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Abbreviations: asRNA, antisense RNA; aTc, anhydrotetracycline; GFP, green fluorescent

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protein; TiP, TetR-inducing peptide.

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Acknowledgments

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We thank the Biomaterial Analysis Center at the Tokyo Institute of Technology for helping us

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with DNA sequencing technique. This work was supported by the Core Research of Evolutional

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Science and Technology program (CREST) at the Japan Science and Technology Agency (JST).

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Author Contributions

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AH performed the experiments and data analysis. AI, YF, and TH assisted with the project. AH

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prepared the initial draft of the manuscript, and AH and TH edited the manuscript.

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Notes

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The authors declare no competing financial interest.

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397

Figure legends

398

Figure 1. The basic design of plasmids used in this study.

399

400

Figure 2. Induction of gfp expression by aTc or nitrate depletion through synthetic gene

401

regulation networks. Anabaena cells were treated with different concentrations of aTc for 24 h

402

under nitrate-replete conditions (+N) or were grown under nitrogen starvation conditions (−N)

403

for 72 h; GFP fluorescence was then measured and normalized (optical density at 750 nm). The

404

experiments using each plasmid were labeled #1 to #5 in the figure. Data represent the mean ±

405

SD (n = 3 from independent cultures).

406

407

Figure 3. Repression of TetR by small asRNA. (A) Dependence of TetR amount on aTc

408

concentrations. Different concentrations of aTc were added to the medium. After 24 h, proteins

409

from each strain grown under +N conditions were extracted, and TetR was detected using a

410

monoclonal anti-TetR antibody by western blotting. (B) Time-course analysis of TetR and GFP

411

amount after the addition of 20 ng/mL aTc. Total proteins from each strain grown under +N

412

conditions were extracted after the indicated time and analyzed by western blotting.

413

414

Figure 4. Induction of GFP in a TetR aptamer-expressing strain. (A) Time-course analysis of

24


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415

GFP amount after the addition of 20 ng/mL aTc when the strain was grown under +N conditions.

416

Total proteins from each strain were extracted after the indicated time and analyzed by western

417

blotting. (B) Time-course analysis of TetR and GFP amount after nitrogen starvation. Total

418

proteins from each strain were extracted after the indicated time and analyzed by western

419

blotting.

420

421

Figure 5. Induction of GFP by aTc and adenine with an adenine off riboswitch. (A) The amount

422

of TetR in strains harboring pCA015 or pCA016 with different concentrations of adenine under

423

+N conditions. Western blotting analysis was performed using a monoclonal anti-TetR antibody.

424

(B) Anabaena strain harboring pCA016 was treated with different concentrations of aTc for 24

425

h under nitrate-replete conditions (+N) or was grown under nitrogen starvation conditions (−N)

426

for 72 h in the absence or presence of 250 µM adenine. GFP fluorescence was then measured

427

and normalized (optical density at 750 nm). Data represent the mean ± SD (n = 3 from

428

independent cultures).

429

430

Figure 6. Induction of GFP under high-light conditions. Expression of GFP was induced under

431

low-light (LL, 30 µmol photons m−2 s−1) or high-light (HL, 150 µmol photons m−2 s−1)

432

conditions when grown in the presence of nitrate. Western blotting analysis was performed

25



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

433

using a polyclonal anti-GFP antibody. (A) Cells harboring pCA015 were grown under low-light

434

conditions. The cells were then irradiated with low or high light intensities, and GFP was

435

induced by aTc for the indicated time. (B) Cells harboring pCA015 or pCA016 were grown

436

under low-light conditions. The cells were then irradiated with low or high light intensities, and

437

GFP was induced by aTc and/or adenine for the indicated time. * indicates that adenine was

438

added 24 h before irradiation with high light intensities.

439

26


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


PpetE

pCA002

PL03

tetR-LVA

PnirA

gfpmut2

pCA013

PL03

tetR-LVA

PL03

gfpmut2 tip-trxAss

PnirA

pCA004

PL03

PnirA

tetR-LVA

gfpmut2

pCA014

PL03

tetR-LVA

PL03

gfpmut2 TetR aptamer

PnirA

pCA011

tetR-LVA

PL03

PnirA

PL03

gfpmut2

pCA015

PL03

tetR-LVA

PL03

gfpmut2

small asRNA for PnirA-tetR

PpetE

pCA012

tetR-LVA

PL03

TetR aptamer with tRNA scaffold

PnirA

PL03

gfpmut2

pCA016

tetR-LVA

small asRNA for PpetE-tetR

PL03

PL03

gfpmut2 adenine off riboswitch

TetR aptamer with tRNA scaffold

FIgure 1



40,000 35,000 GFP fluorescence (au)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

30,000 25,000

Page 28 of 33

aTc 0 20 +N 200 2,000 0 −N (ng/ml)

20,000 15,000 10,000 5,000 0 pCA004 (Control)

#1

pCA011 (small asRNA)

#2

pCA013 (TiP-TrxAss)

#3

pCA014 (TetR aptamer)

pCA015 (TetR aptamer + tRNA scaffold)

#4

#5

Figure 2


Page 29 of 33

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A

pCA004 (Control) aTc

0

2

pCA011 (Small asRNA) 20

200

0

2

20

200

(ng/ml)

anti-TetR

B

pCA004 (Control) Time

0

3

8

24

pCA011 (Small asRNA) 48

72

0

3

8

24

48

72 (h)

anti-TetR

anti-GFP

Figure 3



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 33

A pCA004 (Control) 0

3

8

24

pCA015 (TetR aptamer + tRNA scaffold) 48

72

0

3

8

24

48

72

(h)

anti-GFP

pCA004 (Control)

B

0

3

8

24

pCA015 (TetR aptamer + tRNA scaffold) 48

72

0

3

8

24

48

72

(h)

anti-TetR

anti-GFP

Figure 4


Page 31 of 33

A

pCA015 (TetR aptamer + tRNA scaffold) Adenine

0

2.5

25

250

pCA016 (pCA015 + adenine off riboswitch) 0

2.5

25

250

(µM)

anti-TetR

B

40,000 35,000 GFP fluorescence (au)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


30,000 25,000

aTc 0 20 +N 200 2,000 0 −N (ng/ml)

20,000 15,000 10,000 5,000 0 Adenine

0

250

(µM)

Figure 5



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 33

A pCA015 (TetR aptamer + tRNA scaffold) LL aTc

HL

20 0

24

48

20 72

0

24

200

48

72

0

24

48

(ng/ml) 72

(h)

anti-GFP

B

pCA015 (TetR aptamer + tRNA scaffold)

pCA016 (pCA015 + adenine off riboswitch)

LL aTc

200

0

200

20

0

0

250

250

250*

Adenine 0

HL

200

24

48

72

0

24

48

72

0

24

48

72

0

24

48

72

0

24

48

200

(ng/ml)

250* 72

0

24

48

(µM) 72

(h)

anti-GFP

#1

#2

#3

#4

#5

Figure 6


Page 33 of 33

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