T7 Promoter Circuit in

Letter Cite This: ACS Synth. Biol. XXXX, XXX, XXX−XXX

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Building an Inducible T7 RNA Polymerase/T7 Promoter Circuit in Synechocystis sp. PCC6803 Haojie Jin,*,† Peter Lindblad,‡ and Devaki Bhaya*,† †

Department of Plant Biology, Carnegie Institution for Science, Stanford, California 94305, United States Microbial Chemistry, Department of Chemistry-Ångström, Uppsala University, Box 523, SE 75120, Uppsala, Sweden

‡

ACS Synth. Biol. Downloaded from pubs.acs.org by OCCIDENTAL COLG on 04/03/19. For personal use only.

S Supporting Information *

ABSTRACT: To develop tightly regulated orthogonal gene expression circuits in the photoautotrophic cyanobacterium Synechocystis sp. PCC6803 (Syn6803), we designed a circuit in which a native inducible promoter drives the expression of phage T7 RNA polymerase (T7RNAP). T7RNAP, in turn, specifically recognizes the T7 promoter that is designed to drive GFP expression. In Syn6803, this T7RNAP/T7promoter-GFP circuit produces high GFP fluorescence, which was further enhanced by using mutant T7 promoters. We also tested two orthogonal inducible promoters, Trc1O and L03, but these promoters drive T7RNAP to levels that are toxic in E. coli. Introduction of a protein degradation tag alleviated this problem. However, in Syn6803, these circuits did not function successfully. This highlights the underappreciated fact that similar circuits work with varying efficiencies in different chassis organisms. This lays the groundwork for developing new orthogonally controlled phage RNA polymerase-dependent expression systems in Syn6803. KEYWORDS: Synechocystis sp. PCC6803, T7 RNA polymerase/T7 promoter circuit, orthogonal promoter, nickel 24 inducible promoter

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INTRODUCTION Cyanobacteria represent a diverse, prokaryotic phototrophic phylum that plays a fundamental role in global carbon, nitrogen, and phosphorus cycles. Since photosynthetic activity can help to reduce greenhouse gases and the culture of cyanobacteria in open ponds on a large scale is relatively feasible, there has been investment in the use of Synechocystis sp. PCC6803 (Syn6803) to produce biofuels,1 ethylene,2 and other high value bioproducts.3 Syn6803 has a doubling time of ∼8−12 h and a wellannotated sequenced genome,4 and genetic manipulation is possible via natural transformation or conjugation.5 Varied biochemical techniques, “omics” approaches, and mutant analyses have been used to analyze oxygenic photosynthesis, stress responses, and signal transduction networks6 in Syn6803, but an extensive synthetic biology-based toolkit to enable efficient bioengineering is still lacking. A critical part of using synthetic control of target gene expression, is the use of reliable, controllable, and inducible promoters. These could be native, orthogonal, or synthetic promoters. Native promoters are often part of complex regulatory networks, so it is challenging to engineer them to be independent of host genetic regulation.7 So far, the native promoters used in Syn6803 have not been strong enough to drive high levels of transcript accumulation8 or suffer from other drawbacks,9 so orthogonal promoter systems are urgently needed.10,11 So far, only two orthogonal inducible promoters have been characterized in Syn6803, Trc1O which can be © XXXX American Chemical Society

induced by IPTG (the cell has constitutively expressed Lac repressor12) and the L03 promoter can be induced by anhydrotetracycline (aTc) when the cell has constitutively expressed transcription factor TetR repressor.13,14 Though these promoters are orthogonal, they are still dependent on native RNA polymerase. Since RNA polymerase is constitutively present in cell,15 there is the possibility of leaky control of these promoters, which may vary with each promoter.16 T7 phage RNA polymerase (T7RNAP) is widely used for heterologous protein expression in bacteria.17 It has stringent specificity for its own promoter and mutations in the T7 promoter initiation region can modulate promoter strength.18,19 The T7 promoter and various mutants have been successfully used to express nitrogenase in Klebsiella and E. coli,20,21 which requires expression from multiple operons. The T7RNAP/T7 promoter circuit has also been used to drive gene expression in plant plastids;22 yeast, or mammalian cells.23,24 However, building the T7RNAP/T7 promoter circuit is problematic because of the potential toxicity of T7RNAP.25 An engineered T7RNAP (T7RNAP*) was generated to successfully drive T7 promoter to express nitrogenase under the control of a protein degradation tag.20,26 T7RNAP* has two synonymous mutations and one active site mutation (R632S), which reduces its toxicity20 but also adversely affects polymerase activity (personal Received: December 10, 2018 Published: April 2, 2019 A

DOI: 10.1021/acssynbio.8b00515 ACS Synth. Biol. XXXX, XXX, XXX−XXX

Letter

ACS Synthetic Biology

Figure 1. Schematic of using inducible promoters to build T7RNAP/T7 promoter circuit in Syn6803. The left panel illustrates an orthogonal inducible system which consists of three layers. The first layer is the constitutively expressed repressor (black arrowhead) controlled by a constitutive promoter (Pconstitutive: green). The second layer is the orthogonal inducible promoter (blue) that drives the t7RNAP gene (pink arrowhead); this is the layer which can be either in the ON or OFF state, based on whether a chemical inducer is present or not. In the absence of a chemical inducer, the repressor can bind with the regulator on the inducible promoter to block the gene transcription; when the repressor disassociates from the regulator after chemical inducer binds, the t7RNAP gene is expressed. In the third layer, the translated T7RNAP can drive the T7 promoter to express the gfp gene. In the right panel, is the native inducible system that uses a native inducible promoter (purple). When the inducer is present, it activates the signal pathway for the promoter and T7RNAP is expressed, which in turn drives the T7 promoter to express gfp. Shapes on the left bottom indicate different components or chemicals.

Figure 2. T7RNAP/T7 promoter circuit assays in E. coli and Syn6803. (A) The circuit consists of two operons: an inducible promoter driving the t7RNAP and the T7 promoter driving the gfp. They were assembled in opposite orientations to avoid interference. Two inducible promoters, L03 (blue) and Trc1O (purple), were used in combination with the umuD degradation tag. A construct in which a T7 promoter directly drives gfp but does not have the t7RNAP operon was used as a negative control. The circuits were cloned into the pPMQAK1 plasmid. (B and C) constructs from panel A were transformed and conjugated into E. coli and Syn6803, respectively. GFP fluorescence was assayed using a microplate reader. The bars in panels B and C represent standard error calculated from three biological and technical replicates.

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communication with the Voigt group). There is only one example where the T7RNAP/T7 promoter circuit was used to amplify a weak promoter in the filamentous cyanobacterium Anabaena.27 In this report, we describe the introduction of the T7RNAP/T7 promoter circuit into Syn6803, a unicellular model cyanobacterium, to build a robust expression system.

RESULTS AND DISCUSSION

There are two options for building an inducible system to drive a T7RNAP/T7 promoter circuit in cyanobacterial cells. The first option is to use an orthogonal inducible promoter which is repressed in the presence of a constitutively expressed repressor. B


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Figure 3. Native inducible promoter controlled T7RNAP/T7 promoter circuit assay in Syn6803. (A) Nickel inducible promoter (PnrsB) was directly used to drive t7RNAP. (B) SyNrsB growth under different nickel concentrations from 0 to 60 h. (C) SyNrsB GFP fluorescence 24 h post nickel induction (seven concentrations between 0 and 200 μM). Autofluorescence levels from SyPMQAK1 (Syn6803 bearing pPMQAK1) were subtracted to get values shown in the figure. (D) Western blot test of T7RNAP and GFP from SyNrsB 24 h post different nickel induction. SyPMQAK1 and SyT7 were used as controls. PsbD was used as sample loading control. The bars in B and C represent a standard error calculated from three biological and technical replicates.

strong promoter to drive t7RNAP is toxic to the cell.20 To circumvent this problem, we introduced the protein degradation tag used in Klebsiella (umuD tag from E. coli)20 to control the level of T7RNAP accumulation in the cell. After fusion of the umuD tag, we were able to successfully build constructs pTrc1O and pL03 based on the Trc1O and L03 promoters, respectively (Figure 2A). We also assembled the construct pT7-gfp, which has a T7 promotor driving gfp but without the t7RNAP operon (Figure 2A). This plasmid was used to test any nonspecific effects of the T7 promoter in cells. All three constructs were verified by sequencing and used for further analysis. E. coli strains which bear these constructs were named EcTrc1O (E. coli bearing pTrc1O), EcL03 (E. coli bearing pL03), and EcT7 (E. coli bearing pT7-gfp). They were assayed for fluorescence levels using a microplate reader. EcL03 displayed the highest GFP fluorescence, while EcTrc1O showed intermediate levels, which were significantly higher than EcT7 (this strain did not show any detectable fluorescence, as expected) (Figure 2B). Next, these constructs were conjugated into Syn6803 which generated exconjugants SyTrc1O (Syn6803 bearing pTrc1O), SyL03 (Syn6803 bearing pL03), and SyT7 (Syn6803 bearing pT7-gfp) and GFP fluorescence was assayed. There was no difference between strains with or without the t7RNAP operon; all three exconjugants only exhibited autofluorescence from endogenous chlorophyll (Figure 2C). To examine why GFP fluorescence was not observed in the exconjugants, we measured the transcript and protein levels of T7RNAP. This revealed that both SyTrc1O and SyL03 exhibited high levels of T7RNAP transcripts compared with the control petB (encodes cytochrome b6) (Figure S1A). However, the T7RNAP protein levels were very low (Figure S1B), which may have been the result of the umuD protein degradation tag present in these constructs. When we tested

The repressor binds to the inducible promoter regulator region (OFF status) and blocks the expression of the t7RNAP gene. In the presence of a chemical inducer the repressor disassociates from the inducible promoter, shifting to the ON status such that the downstream gene is transcribed. The translated protein (T7RNAP) can drive transcription from a T7 promoter leading to expression of a reporter gene such as gfp (Figure 1, left panel). The second option is to use a native inducible promoter to drive t7RNAP. The system is OFF under normal growth conditions, but when an inducer is added the system is ON (Figure 1, right panel), leading to an outcome similar to that described above. The advantage of an orthogonal controller system is that it is not likely to interfere with the cellular machinery. Two characterized orthogonal inducible promoters in Syn6803, Trc1O and L03, have been used for the expression of gfp and yfp,12,14 but it is not known whether these promoters can be used to express T7RNAP, since its accumulation can be toxic or lethal to the cell. To test these promoters, we used them in the absence of the repressor, such that the levels of the expressed proteins are constitutive and high. If the cells survive, we would assume that levels of T7RNAP are not toxic to the cell. Two operons which consist of orthogonal inducible promoters (Trc1O and L03) to drive t7RNAP and the compatible T7 promoter driving gf p reporter gene were assembled. To simplify the process, we cloned these two operons into a single replicative plasmid backbone, pPMQAK1, in opposite orientations to eliminate any negative effects (Figure 2A). pPMQAK1 is a plasmid (based on the broad host range plasmid, RSF1010) which can stably exist and propagate in Syn6803.12 The initial attempt to use the Trc1O or L03 promoter to drive t7RNAP was unsuccessful in E. coli, that is, we were unable to get any viable transformants. This is consistent with previously published work which demonstrated that using a C


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Figure 4. Different versions of T7 promoter strength in Syn6803. (A) Constructs were built based on pNrsB which have different versions of T7 promoter to drive the gfp gene. Four mutated T7 promoter versions (mutation sites in red) were tested. (B) Constructs from panel A were conjugated into Syn6803. Exconjugants fluorescence was assayed using a microplate reader 24 h post 40 μM nickel induction. Autofluorescence from SyPMQAK1 was subtracted to get the value shown in the figure. The bars in the histogram figure represent a standard error calculated from three biological and technical replicates.

4A). These constructs were conjugated into Syn6803, which generated SyNrsB-T7mut1 to SyNrsB-T7mut4. GFP fluorescence from the exconjugants was assayed using the microplate reader. The results showed that the different versions of T7 promoters had varying strengths, among which the T7mut2 had over twice the strength of the WT T7 promoter (Figure 4B). In conclusion, we have demonstrated the feasibility of building a T7RNAP/T7 promoter circuit in the unicellular cyanobacterium, Syn6803. We also show that different versions of T7 promoter strengths (Figure 4B) can be generated which may be useful when proteins need to be expressed at different levels. In further iterations, it may be possible to develop a panel of T7 promoters of various strengths.

constructs that did not contain the umuD tag, we found that levels of GFP fluorescence were much higher (Figure S1C). We conclude that although the level of T7RNAP (containing the umuD protein degradation tag) under Trc1O and L03 promoters were high enough to effectively drive the T7 promoter to express GFP in E. coli (Figure 2B), their levels were not high enough to function in Syn6803 (Figure 2C, Figure S1B). To test the hypothesis that T7RNAP levels had to be high enough in Syn6803 to express T7 promoter driving GFP to detectable fluorescence levels, we exploited a native nickel inducible promoter (PnrsB) to drive t7RNAP. PnrsB promoter strength is a function of added nickel, and prior studies have shown that expression of a fluorescence reporter can reach a maximal level when nickel concentration reaches 10 μM.28 Conjugation of pNrsB (shown in Figure 3A) into Syn6803 generated the exconjugant SyNrsB. The growth and GFP fluorescence of SyNrsB were both assayed using a microplate reader under varying nickel concentrations. The results showed that cell growth was totally inhibited when nickel concentration reached 80 μM or higher, and the maximum nickel concentration under which cells maintain growth is 40 μM (Figure 3B). The highest GFP fluorescence was also measured at 40 μM nickel concentration (Figure 3C). Western blots were conducted at the same time to quantify levels of both T7RNAP and GFP, and showed results that were consistent with the microplate reader assay (Figure 3D). Prior studies have demonstrated that mutations in the T7 promoter can affect promoter strength.19 For example, four mutants of the T7 promoter had strengths that varied by 20-fold in Klebsiella.20 We substituted the wild-type (WT) T7 promoter in pNrsB with different versions of mutated T7 promoters, to generate constructs: pNrsB-T7mut1 to pNrsB-T7mut4 (Figure

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MATERIALS AND METHODS Strains Used and Growth Conditions. The unicellular cyanobacterium Syn6803 used in this study is a spontaneous glucose tolerant mutant, which can grow in the dark when BG11 medium is supplemented with glucose (5 mM to 20 mM).29 Here, 5 mM glucose was added to BG11 medium to obtain optimized growth. The strain was grown in BG11 liquid medium at 30 °C with shaking (50 or 250 mL flask, 170 rpm) or on agar plates under 25 μE m−2 s−1 continuous light. Escherichia coli strain DH5α which is grown in Luria−Bertani medium, was used for plasmid cloning, propagation, and GFP assays (37 °C, 200 rpm). Antibiotics were added at the appropriate concentrations: 50 or 100 μg/mL carbenicillin (Cb), and 25 or 50 μg/mL kanamycin (Km) for Syn6803 and E. coli, respectively. E. coli harboring different constructs were grown overnight and then inoculated into fresh LB medium with requisite antibiotics. For strains carrying the pTrc1O or pL03 plasmid, D


Letter


−70 °C until further processing. Total RNA was isolated using Zymo Research Direct-zol RNA MiniPrep Kit (Cat. No. R2050). iScript cDNA Synthesis Kit (Cat. No. 1708891) from Bio-RAD was used for cDNA synthesis, non iScript reverse transcriptase samples were used as negative control. SensiFAST SYBR No-ROX kit from BIOLINE (Cat. No. BIO-98020) was used for qRT-PCR. The qRT-PCRs were performed on Roche LightCycler 480 Multiwell 96 Plate covered with Optical Sealing Foils (Cat. No. 04729749001). petB was selected as a reference gene for analyses.31 The PCR data was analyzed using LinRegPCR software.33 All primers are listed in Table S2. GFP Assay through Microplate Reader. The GFP fluorescence was assayed using the TECAN Infinite M1000 PRO microplate reader. GFP excitation and emission wavelengths are 488 ± 12 and 516 ± 12 nm, respectively; OD600 and OD730 which are indicative of cell density were also measured for E. coli and Syn6803, respectively, for data analysis. For each sample, 120 μL of culture was transferred into a Greiner 96 Flat Bottom Transparent polystyrol microplate (Cat. No: 655101) at the indicated time points. Each construct had three biological and three technical replicates. Cell Extract and Western Blot. For Western blot assay, the whole cell lysate was prepared as described in Xue et al.34 Protein concentration was spectroscopically quantified with the NanoDrop1000, and each well was loaded with 80 μg of total protein. SDS/PAGE was performed using Bio-Rad Precast 12% gel (Cat. 5671044). Cell extracts were incubated with 4× Laemmli Sample Buffer (cat. 1610747) and boiled for 5 min before loading onto the gel. For immunoblot analysis, proteins on SDS/PAGE were transferred onto an Immun-Blot PVDF membrane (cat. 1620177) using the Trans-Blot SD Semi-Dry Transfer Cell (Bio-Rad), probed with anti-T7RNAP primary antibody and donkey anti-goat secondary antibody (Abcam Cat. Ab6885); anti-GFP monoclonal primary antibody (ThermoFisher cat. MA5−15256-HRP) and goat anti-mouse IgG (H+L) secondary antibody (ThermoFisher cat. G-21040); and antipsbD primary antibody (Agrisera cat. AS06 146) and goat antirabbit IgG (H + L) secondary antibody (Bio-Rad cat. 1706515). The protein was visualized by WesternBright ECL (cat. K12045-D20) which is optimized for chemiluminescent Western blots images using X-ray film.

samples were taken at the indicated time points for the microplate reader GFP fluorescence assay. Syn6803 strains bearing the different constructs, were initially grown for 24 h, starting with an initial OD730 = 0.1. The strains containing the pTrc1O or pL03 plasmids, were taken at the indicated time points to test GFP fluorescence; the cells were grown for 46 h for qRT-PCR. For strains bearing pNrsB, the nickel was added at the indicated concentrations, and samples were taken at different time points for the microplate reader assay; Western blot was conducted with cells, 24 h post-nickel-induction. All strains and plasmids are listed in Table S1. Plasmid Construction. All constructs were built using Gibson isothermal assembly.30 All parts which had ∼20 bp overlap with the up and downstream fragments were either amplified using Phusion High-Fidelity DNA Polymerase or synthesized as single-stranded nucleotides from the Stanford PAN facility (when the size was lower than 160 bp). Singlestranded nucleotides from the PAN facility were dissolved into annealing buffer (10 mM Tris, pH 7.5−8.0; 50 mM NaCl; 1 mM EDTA) and annealed to double-stranded DNA based on the Protocol for Annealing Oligonucleotides from Sigma (https:// www.sigmaaldrich.com/technical-documents/protocols/ biology/annealing-oligos.html). DNA fragments were directly synthesized by Integrated DNA Technology (IDT). For construction of theT7RNAP/T7 circuit constructs (pTrc1O, pL03, and pNrsB) the RSF1010-based replicative plasmid backbone was generated by digesting Ptrc1O-GFP12 with EcoRI and PstI. In addition to the plasmid backbone, five fragments were generated to assemble the constructs. The first fragment contains the T7 promoter which was synthesized by Stanford PAN Facility; the second fragment was the GFP fragment which was amplified through primers DB348 and DB557 using Ptrc1O-GFP as a template; the third fragment was the terminator for the t7RNAP gene, which was synthesized from Stanford PAN Facility; the fourth fragment was the t7RNAP open reading frame which was amplified by DB551 and DB324 using total DNA from the BL21 strain as template; the fifth fragment was the synthesized fragment from IDT, which consists of the promoter combined with or without umuD degradation tag. Two orthogonal but fully characterized ribosome binding sites (RBSs) were used to eliminate the influence of the native genetic regulation network.31,28 One from BioBrick [BBa_B0034] (Registry of Standard Biological Parts) for gf p,28 the other (RBS*) is a synthetic RBS which is optimized based on the Syn6803 genome31 for t7RNAP gene. Both RBSs showed low sequence context and stable efficiency from a library of 11 tested RBSs in Syn6803.28 Constructs were verified by sequencing using the following primers: DB122, DB124, DB117, DB41, DB339, DB40, DB551, DB552, DB553, DB206, and DB139. All oligonucleotides are listed in Table S2; all synthesized fragments are in Supplementary 1.1. Conjugation and Syn6803 Exconjugants Selection. Syn6803 conjugation protocol and total DNA isolation were described by Jin et al.32 Total DNA was used as template to amplify the whole circuit by two primer pairs, in two parts, DB138 and DB41 and DB339 and DB139. The amplicons were verified by sequencing using the same primers that were used for amplification. RNA Isolation and Real-Time qRT-PCR To Assay t7RNAP Transcript. All T7RNAP/T7 promoter circuit exconjugants were grown for 46 h in BG11 medium supplemented with required antibiotics starting with an initial OD730 = 0.1. Samples were then collected at 4 °C and stored at

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssynbio.8b00515.

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Plasmids and strains used in this study; oligonucleotides used in this study; synthesized assembly fragments (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Haojie Jin: 0000-0001-7460-4374 Author Contributions

H.J. designed and conducted the experiments and analyzed the results; H.J. and D.B. wrote the manuscript with help from P.L. Notes

The authors declare no competing financial interest. E


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(17) William Studier, F., Rosenberg, A. H., Dunn, J. J., and Dubendorff, J. W. (1990) Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 185, 60−89. (18) Ikeda, R. A., Warshamana, G. S., and Chang, L. L. (1992) In vivo and in vitro activities of point mutants of the bacteriophage T7 RNA polymerase promoter. Biochemistry 31 (37), 9073−9080. (19) Bandwar, R. P., Jia, Y., Stano, N. M., and Patel, S. S. (2002) Kinetic and thermodynamic basis of promoter strength: multiple steps of transcription initiation by T7 RNA polymerase are modulated by the promoter sequence. Biochemistry 41 (11), 3586−3595. (20) Temme, K., Zhao, D., and Voigt, C. A. (2012) Refactoring the nitrogen fixation gene cluster from Klebsiella Oxytoca. Proc. Natl. Acad. Sci. U. S. A. 109 (18), 7085−7090. (21) Wang, X., Yang, J.-G., Chen, L., Wang, J.-L., Cheng, Q., Dixon, R., and Wang, Y.-P. (2013) Using synthetic biology to distinguish and overcome regulatory and functional barriers related to nitrogen fixation. PLoS One 8 (7), No. e68677. (22) McBride, K. E., Schaaf, D. J., Daley, M., and Stalker, D. M. (1994) Controlled expression of plastid transgenes in plants based on a nuclear DNA-encoded and plastid-targeted T7 RNA polymerase. Proc. Natl. Acad. Sci. U. S. A. 91 (15), 7301−7305. (23) Benton, B. M., Eng, W. K., Dunn, J. J., Studier, F. W., Sternglanz, R., and Fisher, P. A. (1990) Signal-mediated import of bacteriophage T7 RNA polymerase into the Saccharomyces Cerevisiae nucleus and specific transcription of target genes. Mol. Cell. Biol. 10 (1), 353−360. (24) Dunn, J. J., Krippl, B., Bernstein, K. E., Westphal, H., and William Studier, F. (1988) Targeting bacteriophage T7 RNA polymerase to the mammalian cell nucleus. Gene 68 (2), 259−266. (25) Temme, K., Hill, R., Segall-Shapiro, T. H., Moser, F., and Voigt, C. A. (2012) Modular control of multiple pathways using engineered orthogonal T7 polymerases. Nucleic Acids Res., 8773. (26) Gonzalez, M., Frank, E. G., Levine, A. S., and Woodgate, R. (1998) Lon-mediated proteolysis of the Escherichia coli UmuD mutagenesis protein: In vitro degradation and identification of residues required for proteolysis. Genes Dev. 12 (24), 3889−3899. (27) Wolk, C. P., Elhai, J., Kuritz, T., and Holland, D. (1993) Amplified expression of a transcriptional pattern formed during development of Anabaena. Mol. Microbiol. 7 (3), 441−445. (28) Englund, E., Liang, F., and Lindberg, P. (2016) Evaluation of promoters and ribosome binding sites for biotechnological applications in the unicellular cyanobacterium Synechocystis sp. PCC 6803. Sci. Rep. 6, 36640. (29) Ikeuchi, M., and Tabata, S. (2001) Synechocystis sp. PCC 6803 - a useful tool in the study of the genetics of cyanobacteria. Photosynth. Res. 70 (1), 73−83. (30) Gibson, D. G., Young, L., Chuang, R.-Y., Venter, J. C., Hutchison Iii, C. A., and Smith, H. O. (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6 (5), 343− 345. (31) Heidorn, T., Camsund, D., Huang, H.-H., Lindberg, P., Oliveira, P., Stensjö , K., and Lindblad, P. (2011) Synthetic biology in cyanobacteria. Methods Enzymol. 497, 539−579. (32) Jin, H., Wang, Y., Idoine, A., and Bhaya, D. (2018) Construction of a shuttle vector using an endogenous plasmid from the cyanobacterium Synechocystis sp. PCC6803. Front. Microbiol., 1662 DOI: 10.3389/fmicb.2018.01662. (33) Ruijter, J. M., Ramakers, C., Hoogaars, W. M. H., Karlen, Y., Bakker, O., van den Hoff, M. J. B., and Moorman, A. F. M. (2009) Amplification efficiency: linking baseline and bias in the analysis of quantitative PCR data. Nucleic Acids Res. 37 (6), e45. (34) Xue, Y., Zhang, Y., Cheng, D., Daddy, S., and He, Q. (2014) Genetically engineering Synechocystis sp. Pasteur Culture Collection 6803 for the sustainable production of the plant secondary metabolite p-Coumaric acid. Proc. Natl. Acad. Sci. U. S. A. 111 (26), 9449−9454.

ACKNOWLEDGMENTS This work was supported by Grant No. MCB-1331151 from the National Science Foundation and the Carnegie Institution for Science. We would like to thank Yonjin Park from the Voigt lab for the helpful input. We thank Arthur Grossman for comments on the paper and members of the Bhaya and Grossman laboratories for helpful discussions. T7 RNA polymerase antisera was generously gifted by Dr. Studier from Brookhaven National Laboratory.

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T7 Promoter Circuit in

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