Research Article pubs.acs.org/synthbio

Orthogonal Ribosome Biofirewall Bin Jia,†,‡ Hao Qi,†,‡ Bing-Zhi Li,†,‡ Shuo Pan,†,‡ Duo Liu,†,‡ Hong Liu,†,‡ Yizhi Cai,§ and Ying-Jin Yuan*,†,‡ †

Key Laboratory of Systems Bioengineering (Ministry of Education), School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, PR China ‡ SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300072, PR China § School of Biological Sciences, University of Edinburgh, Daniel Rutherford Building G.24, The King’s Buildings, Edinburgh EH9 3BF, United Kingdom S Supporting Information *

ABSTRACT: Biocontainment systems are crucial for preventing genetically modified organisms from escaping into natural ecosystems. Here, we describe the orthogonal ribosome biofirewall, which consists of an activation circuit and a degradation circuit. The activation circuit is a genetic AND gate based on activation of the encrypted pathway by the orthogonal ribosome in response to specific environmental signals. The degradation circuit is a genetic NOT gate with an output of I-SceI homing endonuclease, which conditionally degrades the orthogonal ribosome genes. We demonstrate that the activation circuit can be flexibly incorporated into genetic circuits and metabolic pathways for encryption. The plasmid-based encryption of the deoxychromoviridans pathway and the genome-based encryption of lacZ are tightly regulated and can decrease the expression to 7.3% and 7.8%, respectively. We validated the ability of the degradation circuit to decrease the expression levels of the target plasmids and the orthogonal rRNA (O-rRNA) plasmids to 0.8% in lab medium and 0.76% in nonsterile soil medium, respectively. Our orthogonal ribosome biofirewall is a versatile platform that can be useful in biosafety research and in the biotechnology industry. KEYWORDS: biofirewall, orthogonal ribosome, genetically modified organisms, biosafety, biocontainment, synthetic biology

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and construct artificial biosystems with high biosafety on a large scale. Many biocontainment technologies have recently been developed to prevent the release of genetically modified organisms (GMOs) into the environment. Gallagher et al.13 developed engineered riboregulators and nucleases to restrict the viability of E. coli cells to media containing exogenously supplied synthetic small molecules. Cai et al.14 used both transcriptional and recombinational “safeguards” to control essential gene functions of Saccharomyces cerevisiae using estradiol. Mandell et al.15 reconstructed essential enzymes to induce dependence on unnatural amino acids for survival. Clement et al.16 designed “Deadman” and “Passcode” switches with different signal inputs and killing mechanisms to kill bacteria efficiently. These existing biocontainment methods mostly focus on preventing the unintended proliferation of genetically modified microorganisms. However, it has been reported that the organisms can survive the killing mechanism by spontaneous mutagenesis14,15 or horizontal gene transfer,17

ith the development of biotechnology, artificial biological systems have been created for various purposes, such as the production of biochemicals,1 bioenergy,2 and therapeutics.3 Moreover, in the past few years, significant progress has been achieved in genome editing and genome synthesis. Hundreds of genomic mutants have been successfully incorporated into single molecules of genomic DNA by MAGE4,5 and CRISPR.6−8 Millions of base-pairs have been successfully assembled in the chemical synthesis of DNA oligos 9−11 in genome synthesis. With these advanced technologies, artificial biological systems12 with increasing numbers of genes can be designed and constructed. Thus, far, most of these artificial biosystems for controlling gene expression were constructed using regulation elements that were directly compatible with the host. However, the complete compatibility of regulation systems between artificial biosystems and their host can cause many problems. First, the compatible gene regulation system makes the artificial biosystems so transparent to the host that the efficiency of regulation is hindered. From a biosafety perspective, there is significant potential for uncontrolled leakage of the artificial biosystems into nature. Therefore, it is a challenge to design © XXXX American Chemical Society

Received: May 7, 2017 Published: August 7, 2017 A

DOI: 10.1021/acssynbio.7b00148 ACS Synth. Biol. XXXX, XXX, XXX−XXX

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circuit and the degradation circuit together and implemented the biofirewall in laboratory conditions and simulated environmental conditions.

presumably causing biocontainment problems in an open environment or ecosystem. Synthetic orthogonal systems are uncoupled from cellular regulation, which makes their biology more amenable to engineering. The orthogonal ribosome (O-ribosome) specifically translates the orthogonal mRNA (O-mRNA) without cross-talk with the endogenous ribosomes and mRNA. Rackham and Chin18 developed methods for the selection and characterization of O-ribosome:O-mRNA pairs. Chubiz and Rao19 developed a computational method for the rational design of O-ribosomes in bacteria. An and Chin20 created an orthogonal gene expression pathway in Escherichia coli by combining orthogonal transcription by T7 RNA polymerase and translation by an O-ribosome. Orelle et al.21 created the first fully orthogonal ribosome−mRNA system by engineering a hybrid 16S-23S rRNA. O-ribosome systems could be developed to perform novel functions without interfering with native translation, theoretically enabling the construction of a parallel and independent system. Currently, the most common approach to information safety is the password firewall system, which utilizes a unique code for authentication to access a resource. To obtain access to information from a computer, a specific password is needed to activate the system. After one session, the system should log out the user or shut down, which deletes the password and silences the system (Figure 1a).

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RESULTS AND DISCUSSION Design of the Activation Circuit. To develop a new regulatory system independent from the natural expression system, we designed a genetic AND gate23 based on the Oribosome.18−20,24,25 Complementary base pair interactions occur between the Shine-Dalgarno (SD) sequence of mRNA and the anti-Shine-Dalgarno (ASD) sequence of rRNA, guiding the 30S ribosomal subunit to the correct position to initiate translation (Figure S1). Orthogonal Shine-Dalgarno (OSD) sequences are mutant sequences that are not recognized by the host ribosomes. The mRNAs with OSD sequences can be specifically translated by O-ribosomes with mutated ASD sequences that are complementary to the OSD sequence. Therefore, it is necessary to simultaneously input both the OmRNA and the O-rRNA to express the target genes (Figure 2a). The AND gate was constructed with pBAD and pLac promoters as the two inputs to drive the transcription of OrRNA and O-mRNA, respectively. The performance of the AND gate was analyzed under conditions combining different concentrations of arabinose and IPTG. When both 1 mM IPTG and 10 mM arabinose were present, the O-mRNA and O-rRNA were produced, resulting in maximal red fluorescent protein (RFP) fluorescence. When only IPTG or arabinose was present, both the pLac and pBAD promoters in the AND gate showed a leakage response. This result showed that the activation circuit basically performed the function of an AND gate in Boolean logic. Characterization of the Activation Circuit. To characterize our O-ribosome system, we investigated the effect of the O-rRNA on the encrypted pathway by quantitative real-time PCR (qRT-PCR). Because the 3′ end of the 16S rRNA is too short to allow a PCR tag primer for detecting the orthogonal 16S rRNA (O-16S rRNA) specifically (Figure S2), we designed a special loop at nucleotides 81−89 to identify the O-16S rRNA and the host 16S rRNA (Figure 2c). Using the NUPACK software,26 we computationally designed a new DNA loop with a similar secondary structure to the wild-type (WT) 16S rRNA.25 Meanwhile, the free energy of the secondary structure was also close to that of the WT loop (Figure 2c). Nucleotides 81−89 of the WT O-16S rRNA and the modified sequence are AGCUUGCUU and GUGAACACU, respectively. The secondary structure free energy values of the WT loop and modified loop were −7.30 kcal/mol and −6.60 kcal/mol, respectively. Orthogonal PCR experiments (Figure S3) illustrated that these PCR tags were able to distinguish O-16S rRNA from WT-16S rRNA. To investigate the effect of the PCR tag on the activity of the O-ribosomes, the expression level of RFP was measured for the two distinct O-16S rRNAs (Figure 3d). When IPTG and arabinose were both present, the fluorescence/OD values for the WT O-rRNA and modified O-rRNA were 1568 and 1608, respectively. When no inducer (IPTG or arabinose) was present, the fluorescence/OD values for the WT O-rRNA and modified O-rRNA were 48 and 45, respectively. These results demonstrated that the modified O-rRNA preserved the function and activity of WT O-rRNA. This artificial loop can be used to track the O-rRNA specifically without interfering with the normal rRNA function. Using specific PCR tags, we quantitatively investigated the interaction between the expression levels of O-rRNA and of the orthogonal genes. As

Figure 1. Firewall biocontainment system. (a) Password firewall system used for personal computers and information security. The computer utilizes a special code for authentication to log in and clears the password to log out. (b) Inspired by this password firewall system, a biofirewall system was designed for biocontainment and biosafety.

Here, we have developed a biocontainment system called the O-ribosome biofirewall, which consists of an activation circuit and a degradation circuit (Figure 1b). The activation circuit is an AND gate,22,23 which uses O-ribosomes for specific activation of the encrypted pathway. To optimize the orthogonal system, we quantified the interaction between the level of orthogonal rRNA (O-rRNA) and the expression levels of the target genes. We then developed methods for plasmidbased encryption and genome-based encryption by switching the ribosome binding sites (RBSs) of the coding sequences (CDSs) to O-ribosome binding sites (O-RBSs). To prevent the horizontal gene transfer of the O-rRNA plasmids, we designed a degradation circuit under the control of environmental signals to cleave O-rRNA plasmids. The degradation circuit is induced to remove the O-rRNA plasmids in the absence of a specific signal, for example, IPTG. Finally, we integrated the activation B

DOI: 10.1021/acssynbio.7b00148 ACS Synth. Biol. XXXX, XXX, XXX−XXX

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Figure 2. Design of the activation circuit. (a) A schematic of the activation circuit is shown. The AND gate was constructed with pBAD and pLac promoters as the two inputs to drive the transcription of O-rRNA and O-mRNA. (b) Cells (strain EJB002) containing O-rRNA and O-RFP were used in the study. The fluorescent response of this AND gate was measured for 49 combinations of input inductions in the standard context, as displayed on the bottom. The inducer concentrations used were 6.4 × 10 −3, 2.5 × 10 −2, 0.1, 0.4, 1.6, 6.4, and 25 mM IPTG and 6.4 × 10 −3, 2.5 × 10 −2, 0.1, 0.4, 1.6, 6.4, and 25 mM arabinose. (c) Modified loop of the 16S rRNA 81−89 used to generate the orthogonal PCR tag. The WT 16S rRNA 81−89 sequence is AGCUUGCUU, and the modified sequence is GUGAACACU. The secondary structure free energy of the WT loop and modified loop were −7.30 kcal/mol and −6.60 kcal/mol, respectively. The NUPACK software26 was used to design the modified loop. (d) Characterization of the translation efficiency of the modified and WT O-ribosomes (strain EJB002 and EJB010). The Relative Fluorescence Unit (RFU) was the fluorescence per OD600. The OFF-state means the cells were grown without the indicated inducers. The ON-state means the cells were grown with 10 mM Ara and 1 mM IPTG. Cells (strain EJB010) containing the O-RFP plasmid and modified O-rRNA plasmids were cultured for 12 h in 5 mL LB media containing 0, 0.01, 0.1, 1, 10 mM Ara and 1 mM IPTG. (e) The expression level of RFP synthesized by O-ribosomes was analyzed. (f) Total rRNA of O-ribosomes was extracted and quantified by quantitative PCR (see Methods for details).

shown in Figure 2e and 2f, the expression levels of O-RFP and O-rRNA increased with increasing arabinose concentration. In addition, the expression of RFP was strongly associated with the level of O-rRNA. These results demonstrated that high levels of O-rRNA improved the expression of O-RFP. According to these results, the high-copy plasmid pSB1A3 (∼100 copies per cell) was used to enhance the expression of O-rRNA and RFP. Two noteworthy points are the potential leakage and cell toxicity. First, we analyzed the leakiness of O-RBS and OrRNA, respectively. As shown in Figure S4, the fluorescence/ OD absorbance values of EJB000, EJB001 and EJB010 are 17, 24 and 41, respectively. These results show that the O-RBS used in this study has very little interaction with the WT-rRNA, and the pBAD promoter accordingly exhibits very little leakage expression of O-rRNA in Luria−Bertani broth (LB) medium. After background removal, the leakage expression of the orthogonal system accounted for 1.6% of the maximum expression. Second, the overexpression of O-rRNA is

potentially toxic to cells due to competition with native rRNA for free ribosomes or to translation initiation at unwanted locations on native mRNAs.27 We measured the cell growth of strains containing WT-rRNA, O-rRNA and empty vector (pSB1A3). As shown in Figure S5, cells containing either O-rRNA or WT-rRNA exhibited tiny growth defects compared with cells containing empty vector. However, we did not observe any growth defect when expressing OrRNA compared with expressing WT-rRNA. These results demonstrated that the orthogonal ribosome system used throughout this study had very little effect on cellular viability. Encryption for Activation Circuit. As a proof-of-concept for the activation circuit, plasmid-based encryption and decryption were applied to the deoxychromoviridans pathway.28−30 Deoxychromoviridans is synthesized heterogeneously from L-tryptophan, catalyzed sequentially by vioA, vioB and vioE. All three genes were placed in a polycistron under the control of the pLac promoter (Figure 3a). The medium was turned dark green by the expression of VioA-VioB-VioE C

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Figure 3. Encryption of synthetic pathway in plasmid. (a) Biosynthetic pathways for deoxychromoviridans are shown. The products are shown in black, and genes expressed by the engineered system are shown in green. (b) Encryption arithmetic of the biosynthetic pathways for deoxychromoviridans. (c) Characterization of plasmid vioABE encryption. EJB020 is MG1655Z1 containing pJBVioABE; EJB040 is MG1655Z1 containing pJBORibo2 and pJBOVioABE. Cells were cultured for 24 h in LB media containing the indicated inducers and antibiotics. Expression of the vioABE gene caused the cells to turn dark green. Tight repression of the vioABE gene caused the cells to remain colorless in LB media. Quantitative assessment of deoxychromoviridans production. Deoxychromoviridans was extracted,29 and its absorbance was measured (deoxychromoviridans, 650 nm). Data are shown as the fraction of maximum absorbance observed. The absorbance values of EJB020, EJB020 + IPTG, EJB030 and EJB030 + IPTG + Ara were 0.117, 0.156, 0.011 and 0.095, respectively. Error bars are SD (Student t test: n.s., not significant; *P = 0.05, **P = 0.001.).

Figure 4. Encryption of lacZ gene in genome. (a) Encryption arithmetic of the lacZ gene in the bacterial genome. (b) Oligo-mediated replacement of SD sequence, confirmed by Sanger sequencing of genomic PCR amplicon. (c) Characterization of genomic lacZ encryption. EJB060 is MG1655Z1 containing pJBORibo1 and O-lacZ mutation. Cells were grown for 24 h in LB media containing the indicated inducers and antibiotics. LB liquid medium supplemented with IPTG and arabinose was used for serial 10-fold dilution and plating. Expression of the lacZ gene causes cells to turn blue on the surface of agar containing X-gal. Tight repression of the lacZ gene causes cells to remain colorless on the surface of agar containing X-gal. Quantitative assessment of the relative lacZ activity. β-Galactosidase activity was measured using the Yeast β-Galactosidase Assay Kit (Thermo Scientific). Data are reported as the fraction of maximum absorbance observed. The absolute numbers for the absorbance values of EJB041, EJB041 + IPTG, EJB060 and EJB060 + IPTG + Ara were 0.372, 0.491, 0.038 and 0.258, respectively. Error bars are SD (Student t test: n.s., not significant; *P = 0.05, **P = 0.001.).

(pJBVioABE) (Figure 3c). To encrypt this synthetic pathway, the SD sequences of vioA, vioB and vioE were replaced simultaneously with O-SD sequences (pJBOVioABE). The expression of the vioABE genes caused the cells to turn dark green. Tight repression of the vioABE gene causes the cells to remain colorless in LB media. The absorbance values of EJB020 (Table S1), EJB020 + IPTG, EJB030 (Table S1) and EJB030 + IPTG + Ara were 0.117, 0.156, 0.011 and 0.095, respectively. Without IPTG input, the leaky expression of the WT vioABE gene turned the cells dark green, with 74.8% of the maximum absorbance observed (Figure 3c). No pigments were observed in LB media for the strains containing encrypted VioABE.

However, the medium turned dark when the O-rRNA plasmids were transformed into cells containing the target pathway plasmid supplemented with IPTG and Ara. The encryption pathway tightly regulated the expression of vioABE and thus decreased the pigment production to 7.3% of that of the WT vioABE pathway (Figure 3c). The results illustrated that the Oribosome system provides strict control of the synthetic pathway, which will provide an efficient way to control the phenotypes of the encrypted synthetic pathway, even when transformed into other bacteria. To create the activation circuit to control the pathway on the host genome, a genomic encryption method was developed D

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Figure 5. Design of the NOT gate and the degradation circuits. (a) The NOT gate was constructed with the LacI/Plac switch and the cI/Plam repressor module. (b) The dose responses of the engineered cI/Plam NOT gate were measured under the IPTG-inducible Ptac promoter. The inducer concentrations used were 0, 4 × 10 −4,1.6 × 10 −3, 6.4 × 10 −3, 2.5 × 10 −2, 0.1, 0.4, 1.6, 6.4, and 25 mM IPTG. EJB070 (MG1655Z1 containing pJBNOT1 integrated into the genome) was used to perform this study. (c) The engineered degradation circuits (the NOT gate) synthesized I-SceI endonuclease, which digested I-SceI sites on the vector and caused plasmid degradation in vivo. EJB080 (MG1655Z1 containing pJBNOT2 integrated into the genome) was used to perform this study. Degradation of target vector with one I-SceI site and two I-SceI sites, respectively. Single colonies cotransformed with the degradation circuits and the target vector with one and two I-SceI sites were restreaked on LB agar plates to obtain single colonies. Dynamics of the password plasmid stability with the degradation circuits in the OFF-state (d) and the ON-state (e). EJB100 (EJB080 containing pJB2SceI) was grown in LB media containing 1 mM IPTG (d) and 0 mM IPTG (e). The fraction of host cells retaining the target plasmid was calculated from the ratio of bacteria with red fluorescent emission and the total bacteria numbers observed. Fluorescence and phase imaging were obtained using a fluorescence microscope (Olympus CX41). The scale bar is 3.77 μm.

through oligo-mediated replacement,31−33 which has been proven to be effective in multiplex automated genome engineering.4,31,34,35 Specifically, the lacZ gene was encrypted as an example (Figure 4a). The original RBS sequence and the encrypted RBS sequence before the start codon of the host lacZ are CACAGGAAACAGC and TTGTTCCGTCCTCC, respectively. As shown in Figure 4b, the WT RBS sequence can be shifted to the O-RBS sequence precisely according to the design. The phenotypes of genomic lacZ alleles were analyzed by serial 10-fold dilution on LB-X-gal agar plates (Figure 4c). The expression of the lacZ gene turned cells blue on the X-gal agar plates. In contrast, cells with the encrypted lacZ gene remained white on X-gal agar plates containing IPTG but turned blue upon the incorporation of O-rRNA plasmids. Figure 4c shows a quantitative assessment of the relative lacZ. The absolute numbers for the absorbance values of EJB041 (Table S1), EJB041 + IPTG, EJB060 and EJB060 + IPTG + Ara were 0.372, 0.491, 0.038 and 0.258, respectively. For the

WT RBS, the leakage expression of WT lacZ was 75.7% of that of the positive control with IPTG input. For the O-RBS, the background expression of encrypted lacZ was only 7.8% of the WT lacZ. Therefore, our activation circuit exhibited stricter control of genes expression. The high leakiness of the pLac promoter is probably attributable to the WT pLac with a CAP binding site used in this study. CAP binding activates transcription in response to the generation of cAMP in a low glucose environment,36 such as LB medium. This biocontainment system integrated O-ribosomes and OmRNAs to generate the output response of genetically modified phenotypes in an encryption-decryption process. In plasmidbased encryption, the orthogonal genes of the pathway could be quickly reassembled by the Gibson method or yeast assembly method. In the genome-based encryption, O-RBSs could be explicitly incorporated into the genome by oligo-media replacement or the CRISPR-Cas9 System.5,37 This encryption E

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Figure 6. Integration of the activation circuit and the degradation circuit. (a) Integration of the activation circuit and the degradation circuit in living cells. The degradation circuit was first integrated into the genome of host cells, and the encrypted deoxychromoviridans pathway and the O-rRNA plasmids were then transformed into the host cells in the presence of 1 mM IPTG and 10 mM Ara. (b) Single colonies (EJB110) were inoculated into 20 mL of LB-IA medium (LB containing 1 mM and 10 mM Ara) at 37 °C for 48 h. Cultures were diluted and plated on LB-IA agar containing Kan, Cm and Amp. Ten randomly picked colonies were used to detect O-rRNA by PCR analysis (red arrows indicate primers labeled P5 and P6), and analysis of the PCR products by electrophoresis on a 1% agarose gel. (c) Single colonies (EJB110) were inoculated in 20 mL of nonsterile soli medium at 37 °C for 48 h. Cultures were diluted appropriately and plated on LB agar containing Kan and Cm. Ten random colonies were picked to detect O-rRNA plasmids in vivo by PCR analysis.

pSB1A3 vector with two I-SceI sites was used as the O-rRNA vector. In addition, to quantify the dynamics of O-rRNA plasmid stability with the degradation circuits in the OFF-state (+ IPTG) and the ON-state (- IPTG), the fraction of host cells retaining the target plasmid (pSB1A3 with 2 × I-SceI sites) was calculated from the ratio of bacteria with red fluorescent emission and the total number of bacteria observed. As shown in Figure 5d, the target plasmid maintained high stability when IPTG was added to inactivate this NOT ate. In contrast, the target plasmid expression decreased to 0.8% after 36 h of degradation (Figure 5e). These experiments suggested that the target vectors exhibited high genetic stability in the presence of IPTG but were dramatically degraded upon the withdrawal of IPTG. This degradation circuit generated NOT gate logic behavior by the control of I-SceI homing endonuclease expression, which could be used to prevent the horizontal gene transfer of target plasmids. Integration of the Activation Circuit and the Degradation Circuit. To prove the concept of the biofirewall with a designed function, the activation circuit and the degradation circuit were integrated in E. coli. The degradation circuit was previously incorporated into the genome of the host cells to obtain the strain EJB080. The encrypted deoxychromoviridans pathway and the O-rRNA plasmids with 2 × SceI sites were simultaneously transformed into EJB080 (Figure 6a).

approach could theoretically be used for biocontainment control on a larger scale. Design of the Degradation Circuit. To avoid the horizontal transfer of O-rRNA plasmids, we designed a genetic NOT gate to cleave O-rRNA plasmids automatically in response to environmental signals. This NOT gate was based on the LacI/pTac switch and CI/pR switch module consisting of lambda gene cI and its regulatory pR promoter. To characterize the NOT gate performance, RFP was first used to profile the output of the pR promoter in this NOT gate. Figure 5b shows that the pR promoter was significantly inhibited by the repressive effect of IPTG (>0.1 mM) on CI and significantly inducible when IPTG was withdrawn. To digest O-rRNA plasmids exclusively, the I-SceI homing endonuclease38,39 was applied. This endonuclease specifically recognizes the 18-bp sequence TAGGGATAACAGGGTAAT, which does not occur in the E. coli genome and thus in theory should do no harm to the host cell. To implement the degradation circuit, RFP was replaced by the I-SceI homing endonuclease in the NOT gate (Figure 5c). To optimize the degradation of OrRNA, two versions of the pSB1A3 vector with 1 × I-SceI site and 2 × I-SceI sites were tested. The degradation circuit was first integrated into the genome of MG1655Z1. The cleavage on the vector with two I-SceI sites was more efficient than that on the vector with one I-SceI site (Figure 5c). Therefore, the F

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expression of vioABE, decreasing the pigment production to 7.3% of the level in the WT vioABE pathway, and the expression of encrypted lacZ was decreased to 7.8% of the WT lacZ level. Fourth, our system was more stable because the degradation circuit conditionally digested the O-rRNA plasmid, permanently silencing the expression of the target pathway. Compared with other killing switches,13−15 the O-ribosome biofirewall caused no harm to the host, potentially reducing the risks of unintended proliferation enabled by spontaneous mutagenesis. Finally, in principle, the regulation of those genetic circuits can be extended to respond to different signals due to the modularity of the design. For example, the aTcinduced pTet promoter could replace the pBAD or pTac promoter of our circuits. Our system could be improved to conditionally degrade the plasmid containing the encrypted pathway by introducing two of the I-SceI sites into this plasmid backbone. Overall, the O-ribosome biofirewall is a highly efficient, flexible and cost-effective platform for biocontainment. We anticipate the widespread use of the O-ribosome biofirewall for ecosystem safety in the biotechnology industry.

To simulate an open environment, we used nonsterile soil medium and examined the microbes present before culturing the biofirewall strains (Figure S6). Microbe colonies of different colors and sizes on LB agar and LB-Amp agar indicated the presence of ampicillin-resistant microbes in the soil, while those microbes were sensitive to kanamycin and chloramphenicol. Subsequently, the performance of biofirewall strains in the laboratory environment and in a simulated open environment (nonsterile soil medium) were investigated separately. As shown in Figure 6b and 6c, almost all the colonies produced dark pigments in LB media with arabinose and IPTG, while no dark pigments were observed in nonsterile soil medium. PCR analysis of those colonies showed that the O-rRNA plasmids were stable in the laboratory environment, indicating that the degradation circuit was regulated tightly in the presence of IPTG. PCR analysis of those colonies showed that the O-rRNA plasmids were degraded in the simulated open environment, implying that there was insufficient environmental IPTG or lactose to repress the degradation circuit, and thus the I-SceI endonuclease digested the O-rRNA plasmids. In general, even though lactose and arabinose are readily found in the environment, most microbes can use them as carbon sources for cell growth, which reduces their concentrations below the essential inducible concentration for the circuits. Therefore, it is reasonable to believe that the degradation circuit would functionally cleave the O-rRNA plasmids in the open environment. The O-rRNA plasmid might still escape to the environment without plasmid cleavage, when some cells are lysed or when endonuclease expression is deactivated by random mutation. Therefore, quantitative PCR was used to examine the presence of O-rRNA plasmids in the cell culture over long periods of cell cultivation. Figure S7 shows the changes in relative copy number of the target plasmids when the biofirewall strains were cultured in LB-IPTG-Ara medium and nonsterile soil medium. The O-rRNA plasmids in the nonsterile soil medium decreased to nearly 0.76% compared with those in the laboratory medium in 48 h. The O-rRNA plasmids remained stable during longterm cultivation when the degradation circuit was tightly repressed by IPTG but were dramatically diminished in the nonsterile soil medium. There are several reasons for this difference. First, cells escaping to the environment grow slower than cells in the laboratory medium due to poor nutrition, leading to smaller amounts of O-rRNA plasmid in the soil medium than in the laboratory medium. Second, most O-rRNA plasmids in the nonsterile soil medium were digested by the ISceI endonuclease expressed in the absence of sufficient environmental IPTG or lactose. Last, the escaping O-rRNA plasmids might be degraded by DNases or microbes (Figure S6) in the soil.40 The O-ribosome biofirewall, as described here, represents the state of the art for microbial biocontainment and offers certain advantages. First, using the central role of Watson−Crick base pairing, we can readily modify the biofirewall by computational modeling and experimental characterization. It is reasonable to choose different O-SD:O-ASD pairs for different target pathways, which will improve the diversity and security of the biofirewall. Second, due to the small size of RBSs, the O-RBS could be incorporated into genetic circuits and metabolic pathways, enabling flexible engineering. Third, the O-RBS and O-rRNA in this work provide a restrictive control, enabling the efficient construction of complex synthetic biological systems. Here, the pathway-encryption method tightly regulated

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METHODS AND MATERIALS Strains and Media. Plasmid cloning work and circuit construct characterization were all performed in the Escherichia coli strain DH10B {F-mcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 ΔlacX74 recA1 endA1 araΔ139 Δara, leu) 7697 galU galK λ-rpsL (StrR) nupG}. MG1655Z1 (F-, λ-, SpR, lacR, tetR), a gift from Michael B Elowitz.41 Cells were cultured in Luria−Bertani broth (LB) media (10 g/L peptone, 5 g/L NaCl, 5 g/L yeast extract). The nonsterile soil medium consisted of 100 g of fresh garden soil and 900 mL of fresh lake water (Tianjin, postcode 300072). Kanamycin (Kan, 50 mg/ mL), ampicillin (Amp, 100 mg/mL) and chloramphenicol (Cm, 34 mg/mL) were added as appropriate. Two inducers were obtained from Sigma-Aldrich: arabinose (Ara) and isopropyl βD-1-thiogalactopyranoside (IPTG). Plasmid Circuit Construction. All plasmids were constructed using standard molecular cloning techniques and Gibson assembly. All plasmid maps are shown in the Supporting Information. Restriction endonucleases, T4 DNA Ligase and Phusion PCR kits from New England BioLabs (NEB) were used. PCR was performed with an ABI Thermal Cycler. Primers were synthesized by Genewiz. All plasmids were transformed into E. coli strain DH10B with standard protocols and isolated with TIANprep Mini Plasmid Kits. Plasmid constructs were verified by restriction digests and sequencing by Genewiz. Details are presented in Supporting Information S4−12, with plasmid maps describing representative circuit constructs. The plasmids pSB1A3, pSB3C5 and pSB4K5 were from the Registry of Standard Biological Parts (http://partsregistry.org). The yeast/E. coli shuttle vector pJB413C was constructed using multichange isothermal mutagenesis by replacing the beta lactamase (bla) gene and Ori site of pRS413 with the CmR gene and p15A ori (Supporting Information Figure S6). Specifically, pRS413 was amplified by PCR with primer pairs that produce two PCR products with homologous ends. After gel purification, the two DNA fragments were assembled by Gibson assembly.42 Pathway Encryption in Plasmid. The pJB413C plasmid was linearized by SacI and BamHI. The WT pJBVioABE (Supporting Information Figure S7) was assembled by transforming linear pJB413C and the PCR products of SDG

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Research Article

ACS Synthetic Biology

Single colonies of EJB080 were used to inoculate LB (1 mL) containing kanamycin and 1 mM IPTG, and the resulting liquid cultures were grown in a shaking incubator for 3 h at 37 °C and 250 rpm. until OD600 0.5−0.6. This culture was used to prepare electrocompetent cells. Two of the RFP target plasmids (pJB1SceI and pJB2SceI) were transformed into electrocompetent cells (EJB080), plated onto LB agar (ampicillin, kanamycin and 1 mM IPTG) and grown for 12 h at 37 °C. Single colonies (EJB090 and EJB100) were used to inoculate 1 mL of LB (ampicillin, kanamycin and 1 mM IPTG) and grown in a shaking incubator at 37 °C until OD600 0.5−0.6. The cultures were spun down at 12 000g, washed once with fresh LB (1 mL) and back-diluted to OD600 0.01 in 20 mL of LB containing 1 mM IPTG for the OFF-state and 0 mM IPTG for ON-state, respectively. RFP fluorescence imaging and phage imaging were then obtained from this cell lawn using a fluorescence microscope (Olympus CX41). The fraction of host cells retaining the target plasmid (Target+) was calculated from the ratio of bacteria exhibiting red fluorescent emission to the total bacteria numbers observed. O-rRNA Plasmid Degradation Assay. The O-rRNA plasmids (pJB2SORibo2) and pJBOVioABE were transformed into electrocompetent cells (EJB080), plated onto LB agar (ampicillin, kanamycin, chloramphenicol and 1 mM IPTG) and grown for 12 h at 37 °C. In the laboratory environment, single colonies (EJB110) were used to inoculate 20 mL of LB medium (ampicillin, kanamycin, chloramphenicol and 1 mM IPTG) and grown in a shaking incubator at 37 °C. Cultures were diluted appropriately after 48 h, plated onto LB agar (ampicillin, kanamycin, chloramphenicol and 1 mM IPTG) and grown at 37 °C. For the open environment, single colonies (EJB110) were used to inoculate 20 mL of nonsterile soil medium and grown in a shaking incubator at 37 °C. Cultures were diluted appropriately after 48 h, plated onto LB agar (kanamycin, chloramphenicol) and grown at 37 °C. P5 (5′TGCCACCTGACGTCTAAGAA-3′) and P6 (5′-TCGCCACCGTCGGTCGCAATG-3′) were used for PCR analysis of the O-rRNA plasmids. To examine the presence of O-rRNA plasmids in the cell culture over a long period of cell cultivation, 1 mL sterile cultures (12 h, 24 h, 36 h, 48 h, 60 h) from both of the media described above were collected by centrifugation (12000 rpm, 5 min) and then filtrated through a 0.22 μm aqueous membrane, respectively. The O-rRNA plasmids were also detected by PCR analysis (P5 and P6) and electrophoresis on a 1% agarose gel. The primers P7 (5′-GATAACGAGCTCCTGCACTG-3′) and P8 (5′-ACTGTGAGCCAGAGTTGCCC-3′) were used to target the chromosomal lacZ as reference gene.

VioA, SD-VioB, and SD-VioC into yeast. The encryption pathway of pJBOVioABE was assembled by transforming linear pJB413C and the PCR products of OSD-VioA, OSD-VioB, and OSD-VioC into yeast. Yeast transformants were combined into a single pool for plasmid recovery into E. coli DH10B, where blue/white screening distinguished empty vector from clones in which the insets were assembled. Colony PCR was used to identify clones encoding full-length inserts to be sent for sequencing (see RADOM43). Pathway Encryption in Genome. Strain MG1655Z1 containing pKD4644 was grown at 32 °C to mid log (OD600 = 0.4−0.6). λ-Red was induced with the addition of 10 mM Larabinose and incubated for 1 h, then quick-chilled in ice for 10 min. To prepare for electroporation, 1 mL of cells was washed twice with cold sterile ddH2O and finally concentrated 20-fold in 50 μL of cold sterile ddH2O. To modify the genome via electroporation, 1 μM oligo 5′-GTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCATTGTTCCGTCCTCCATGACCATGATTACGGATTCACTGGCCGTCGTTTTACA-3′ was mixed into 50 μL electrocompetent cells and placed in a Bio-Rad electroporator (0.1 cm cuvette, 1.80 kV). After electroporation, cells were allowed to recover for 3 h in LB before plating for blue/white screening. β-Galactosidase activity was measured using the Yeast β-Galactosidase Assay Kit (Thermo Scientific). Relative Fluorescence Measurements. Single colonies were used to inoculate LB (5 mL) overnight in a shaking incubator at 37 °C and 220 rpm. Cultures were back-diluted to OD600 0.5−0.6 into LB (5 mL) containing appropriate antibiotics and inducers. The cultures were then grown in a shaking incubator for 8 h at 37 °C and 220 rpm. Then, 200 μL cell culture was transferred to a 96-well fluorescence plate, and the optical density was read at 600 nm. RFP fluorescence measurements were performed on a Spectramax M2 (Molecular Devices) with excitation at 584 nm and measurement of the emission at 617 nm. The experiment was performed in triplicate. The Fluorescence/OD was the fluorescence per OD600. The Relative Fluorescence/OD is reported as the fraction of the maximum Fluorescence/OD. Quantitative Real-Time PCR. To measure the performance of the O-ribosomes at the RNA level, the relative rRNA concentrations were measured with quantitative PCR. We used the relative gene expression (the 2-ΔΔCt method45) to present the data of the O-16S rRNA relative to the internal control gene the WT-23S rRNA. The growth conditions for the collection of total RNA were as described above. Total RNA was extracted with the RNeasy Protect Bacteria Mini Kit (Qiagen), according to the manufacturer’s instructions. Firststrand cDNA was synthesized with the SuperScript III FirstStrand Synthesis System for RT-PCR (Invitrogen). Random hexamers were used for reverse transcription. Fast SYBR Green Master Mix (Applied Biosystems) was used for real-time PCR, and experiments were performed on the StepOnePlus RealTime PCR System (Applied Biosystems). The target primers of O-16S rRNA were P1 (5′-TAACAGGAAGAGTGAACACT3′) and P2 (5′-GCCTAGGTGAGCCGTTACCC-3′), and the reference primers of WT-23S rRNA were P3 (5′-CTAAGCGTACACGGTGGATG-3′) and P4 (5′-CCTCGGGGTACTTAGATGTT-3′). The relative O-rRNA lever is reported as the fold changes of the background O-rRNA lever. Fluorescent-Based Plasmids Degradation Assay. The degradation circuit was linearized by ApaI and integrated into the genome of MG1655Z1 by λ-red recombination44 (EJB080).

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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.7b00148. Table S1, Supplementary figures (Figure S1−S7), plasmid maps (Figure S8−S15) (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Fax: 86-22-27403888. E-mail: [email protected]. ORCID

Yizhi Cai: 0000-0003-1663-2865 Ying-Jin Yuan: 0000-0003-0553-0089 H

DOI: 10.1021/acssynbio.7b00148 ACS Synth. Biol. XXXX, XXX, XXX−XXX

Research Article

ACS Synthetic Biology Author Contributions

(10) Annaluru, N., Muller, H., and Mitchell, L. (2014) Total Synthesis of a Functional Designer Eukaryotic Chromosome. Science 344, 55−58. (11) Dymond, J. S., Richardson, S. M., Coombes, C. E., Babatz, T., Muller, H., Annaluru, N., Blake, W. J., Schwerzmann, J. W., Dai, J., Lindstrom, D. L., Boeke, A. C., Gottschling, D. E., Chandrasegaran, S., Bader, J. S., and Boeke, J. D. (2011) Synthetic chromosome arms function in yeast and generate phenotypic diversity by design. Nature 477, 471−476. (12) Nandagopal, N., and Elowitz, M. B. (2011) Synthetic biology: integrated gene circuits. Science 333, 1244−8. (13) Gallagher, R. R., Patel, J. R., Interiano, A. L., Rovner, A. J., and Isaacs, F. J. (2015) Multilayered genetic safeguards limit growth of microorganisms to defined environments. Nucleic Acids Res. 43, 1945− 54. (14) Cai, Y., Agmon, N., Choi, W. J., Ubide, A., Stracquadanio, G., Caravelli, K., Hao, H., Bader, J. S., and Boeke, J. D. (2015) Intrinsic biocontainment: multiplex genome safeguards combine transcriptional and recombinational control of essential yeast genes. Proc. Natl. Acad. Sci. U. S. A. 112, 1803−8. (15) Mandell, D. J., Lajoie, M. J., Mee, M. T., Takeuchi, R., Kuznetsov, G., Norville, J. E., Gregg, C. J., Stoddard, B. L., and Church, G. M. (2015) Biocontainment of genetically modified organisms by synthetic protein design. Nature 518, 55−60. (16) Chan, C. T. Y., Lee, J. W., Cameron, D. E., Bashor, C. J., and Collins, J. J. (2015) "Deadman" and "Passcode" microbial kill switches for bacterial containment. Nat. Chem. Biol. 12, 1−7. (17) Wright, O., Delmans, M., Stan, G. B., and Ellis, T. (2015) GeneGuard: A modular plasmid system designed for biosafety. ACS Synth. Biol. 4, 307−316. (18) Rackham, O., and Chin, J. W. (2005) A network of orthogonal ribosome x mRNA pairs. Nat. Chem. Biol. 1, 159−166. (19) Chubiz, L. M., and Rao, C. V. (2008) Computational design of orthogonal ribosomes. Nucleic Acids Res. 36, 4038−4046. (20) An, W., and Chin, J. W. (2009) Synthesis of orthogonal transcription-translation networks. Proc. Natl. Acad. Sci. U. S. A. 106, 8477−8482. (21) Orelle, C., Carlson, E. D., Szal, T., Florin, T., Jewett, M. C., and Mankin, A. S. (2015) Protein synthesis by ribosomes with tethered subunits. Nature 524, 119. (22) Wang, B., Kitney, R. I., Joly, N., and Buck, M. (2011) Engineering modular and orthogonal genetic logic gates for robust digital-like synthetic biology. Nat. Commun. 2, 508. (23) Moon, T. S., Lou, C., Tamsir, A., Stanton, B. C., and Voigt, C. A. (2012) Genetic programs constructed from layered logic gates in single cells. Nature 491, 249−253. (24) Rackham, O., and Chin, J. W. (2005) Cellular logic with orthogonal ribosomes. J. Am. Chem. Soc. 127, 17584−5. (25) Barrett, O. P., and Chin, J. W. (2010) Evolved orthogonal ribosome purification for in vitro characterization. Nucleic Acids Res. 38, 2682−2691. (26) Zadeh, J. N., Steenberg, C. D., Bois, J. S., Wolfe, B. R., Pierce, M. B., Khan, A. R., Dirks, R. M., and Pierce, N. A. (2011) NUPACK: Analysis and design of nucleic acid systems. J. Comput. Chem. 32, 170− 173. (27) Yang, C., Hockenberry, A. J., Jewett, M. C., and Amaral, L. A. N. (2016) Depletion of Shine-Dalgarno Sequences within Bacterial Coding Regions Is Expression Dependent. G3: Genes, Genomes, Genet. X, 1−9. (28) Torella, J. P., Boehm, C. R., Lienert, F., Chen, J.-H., Way, J. C., and Silver, P. a. (2014) Rapid construction of insulated genetic circuits via synthetic sequence-guided isothermal assembly. Nucleic Acids Res. 42, 681−9. (29) 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. 40, 8773−8781. (30) Lee, M. E., Aswani, A., Han, A. S., Tomlin, C. J., and Dueber, J. E. (2013) Expression-level optimization of a multi-enzyme pathway in

B.J. was the first author and designed most of the experiments; B.J., S.P., D.L., H.L. and Y.-Z.C. performed the research; B.J. and Y.-Z.C. designed and constructed the genetic circuits, and S.P. and D.L. assembled the vioABE pathways. H.L. detected and analyzed the products of the vioABE pathways. B.J., H.Q., and B.-Z.L. wrote the manuscript; Y.-J.Y. and Y.-Z.C. edited the manuscript; and all the work described was guided by Y.-J.Y. Notes

The authors declare no competing financial interest. The plasmids produced in this work may be obtained from the SynbioML repository by request at http://www.synbioml.org/ ModuleLib/index.jsp.

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ACKNOWLEDGMENTS This work was funded by the Ministry of Science and Technology of China (“973” Program, 2014CB745100), the International S&T Cooperation Program of China (2015DFA00960), and the National Natural Science Foundation of China (21390203 and 21621004). We thank the Tianjin University 2012 iGEM team for their initial work and ideas on the O-Key system. We thank Michael B. Elowitz for providing the MG1655Z1 strains.

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ABBREVIATIONS RBS, ribosome binding sites; SD, Shine-Dalgarno; ASD, AntiShine-Dalgarno; CDS, coding sequence; PCR, polymerase chain reaction; bp, base pair; kb, kilobase pair; rRNA, ribosomal RNA; O-rRNA, orthogonal rRNA; WT, wild type; O-, orthogonal-; Kan, kanamycin; Amp, ampicillin; Cm, chloramphenicol

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REFERENCES

(1) Smanski, M. J., Zhou, H., Claesen, J., Shen, B., Fischbach, M. A., and Voigt, C. A. (2016) Synthetic biology to access and expand nature’s chemical diversity. Nat. Rev. Microbiol. 14, 135−149. (2) Atsumi, S., Hanai, T., and Liao, J. C. (2008) Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels. Nature 451, 86−9. (3) Lienert, F., Lohmueller, J. J., Garg, A., and Silver, P. a. (2014) Synthetic biology in mammalian cells: next generation research tools and therapeutics. Nat. Rev. Mol. Cell Biol. 15, 95−107. (4) Wang, H. H., Kim, H., Cong, L., Jeong, J., Bang, D., and Church, G. M. (2012) Genome-scale promoter engineering by coselection MAGE. Nat. Methods 9, 591−593. (5) Ronda, C., Pedersen, L. E., Sommer, M. O. A., and Nielsen, A. T. (2016) CRMAGE: CRISPR Optimized MAGE Recombineering. Sci. Rep. 6, 19452. (6) Konermann, S., Brigham, M. D., Trevino, A. E., Joung, J., Abudayyeh, O. O., Barcena, C., Hsu, P. D., Habib, N., Gootenberg, J. S., Nishimasu, H., Nureki, O., and Zhang, F. (2014) Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature 517, 2−11. (7) DiCarlo, J. E., Chavez, A., Dietz, S. L., Esvelt, K. M., and Church, G. M. (2015) Safeguarding CRISPR-Cas9 gene drives in yeast. Nat. Biotechnol. 33, 1250−1255. (8) Caliando, B. J., and Voigt, C. A. (2015) Targeted DNA degradation using a CRISPR device stably carried in the host genome. Nat. Commun. 6, 6989. (9) Ostrov, N., Landon, M., Guell, M., Kuznetsov, G., Teramoto, J., Cervantes, N., Zhou, M., Singh, K., Napolitano, M. G., Moosburner, M., Shrock, E., Pruitt, B. W., Conway, N., Goodman, D. B., Gardner, C. L., Tyree, G., Gonzales, A., Wanner, B. L., Norville, J. E., Lajoie, M. J., and Church, G. M. (2016) Design, synthesis, and testing toward a 57-codon genome. Science 353, 819−822. I

DOI: 10.1021/acssynbio.7b00148 ACS Synth. Biol. XXXX, XXX, XXX−XXX

Research Article

ACS Synthetic Biology the absence of a high-throughput assay. Nucleic Acids Res. 41, 10668− 78. (31) Dicarlo, J. E., Conley, A. J., Penttilä, M., Jäntti, J., Wang, H. H., and Church, G. M. (2013) Yeast oligo-mediated genome engineering (YOGE). ACS Synth. Biol. 2, 741−749. (32) Wang, H. H., Xu, G., Vonner, A. J., and Church, G. (2011) Modified bases enable high-efficiency oligonucleotide-mediated allelic replacement via mismatch repair evasion. Nucleic Acids Res. 39, 7336− 47. (33) Li, Y., Gu, Q., Lin, Z., Wang, Z., Chen, T., and Zhao, X. (2013) Multiplex Iterative Plasmid Engineering for combinatorial optimization of metabolic pathways and diversification of protein coding sequences. ACS Synth. Biol. 2, 651. (34) Isaacs, F. J., Carr, P. A., Wang, H. H., Lajoie, M. J., Sterling, B., Kraal, L., Tolonen, A. C., Gianoulis, T. A., Goodman, D. B., Reppas, N. B., Emig, C. J., Bang, D., Hwang, S. J., Jewett, M. C., Jacobson, J. M., and Church, G. M. (2011) Precise manipulation of chromosomes in vivo enables genome-wide codon replacement. Science 333, 348−353. (35) Wang, H. H., Isaacs, F. J., Carr, P. A., Sun, Z. Z., Xu, G., Forest, C. R., and Church, G. M. (2009) Programming cells by multiplex genome engineering and accelerated evolution. Nature 460, 894−898. (36) Yu, X.-M., and Reznikoff, W. S. (1984) Deletion analysis of the CAP-cAMP binding site of the Escherichia coli lactose promoter. Nucleic Acids Res. 12, 5449−5464. (37) Li, Y., Lin, Z., Huang, C., Zhang, Y., Wang, Z., Tang, Y. J., Chen, T., and Zhao, X. (2015) Metabolic engineering of Escherichia coli using CRISPR-Cas9 meditated genome editing. Metab. Eng. 31, 13− 21. (38) Lin, Z., Xu, Z., Li, Y., Wang, Z., Chen, T., and Zhao, X. (2014) Metabolic engineering of Escherichia coli for the production of riboflavin. Microb. Cell Fact. 13, 104. (39) Mitchell, L. a., and Boeke, J. D. (2014) Circular permutation of a synthetic eukaryotic chromosome with the telomerator. Proc. Natl. Acad. Sci. U. S. A. 111, 17003. (40) Nielsen, K. M., Johnsen, P. J., Bensasson, D., and Daffonchio, D. (2007) Release and persistence of extracellular DNA in the environment. Environ. Biosaf. Res. 6, 37−53. (41) Cox, R. S., 3rd, Dunlop, M. J., and Elowitz, M. B. (2010) A synthetic three-color scaffold for monitoring genetic regulation and noise. J. Biol. Eng. 4, 10. (42) Gibson, D. G., Young, L., Chuang, R. Y., Venter, J. C., Hutchison, C. A., 3rd, and Smith, H. O. (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343− 345. (43) Lin, Q., Jia, B., Mitchell, L. A., Luo, J., Yang, K., Zeller, K. I., Zhang, W., Xu, Z., Stracquadanio, G., Bader, J. S., and Boeke, J. D. (2014) RADOM, an Efficient In Vivo Method for Assembling Designed DNA Fragments up to 10 kb Long in Saccharomyces cerevisiae. ACS Synth. Biol. 4, 213−20. (44) Datsenko, K. A., and Wanner, B. L. (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. U. S. A. 97, 6640−6645. (45) Schmittgen, T. D., and Livak, K. J. (2008) Analyzing real-time PCR data by the comparative CT method. Nat. Protoc. 3, 1101−1108.

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DOI: 10.1021/acssynbio.7b00148 ACS Synth. Biol. XXXX, XXX, XXX−XXX