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Orthogonal ribosome bio-firewall Bin Jia, Hao Qi, Bing-Zhi Li, Shuo Pan, Duo Liu, Hong Liu, Yizhi Cai, and Ying-Jin Yuan ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.7b00148 • Publication Date (Web): 07 Aug 2017 Downloaded from http://pubs.acs.org on August 8, 2017

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ACS Synthetic Biology

Orthogonal ribosome bio-firewall

1 2

Bin Jia†,‡, Hao Qi†,‡, Bing-Zhi Li†,‡, Shuo Pan†,‡, Duo Liu†,‡, Hong Liu†,‡, Yizhi Cai§,

3

and Ying-Jin Yuan* †,‡

4 5

†

6

Technology, Tianjin University, Tianjin, 300072, P. R. China

7

‡

8

Tianjin, 300072, P. R. China

9

§

10

Key Laboratory of Systems Bioengineering (Ministry of Education), School of Chemical Engineering and

SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin),

Daniel Rutherford Building G.24, School of Biological Sciences, University of Edinburgh, The King’s Buildings,

Edinburgh EH9 3BF, United Kingdom

11 12

ABSTRACT

13

Biocontainment systems are crucial for preventing genetically modified organisms

14

from escaping into natural ecosystems. Here, we describe the orthogonal ribosome

15

bio-firewall, which consists of an activation circuit and a degradation circuit. The

16

activation circuit is a genetic AND gate based on activation of the encrypted pathway

17

by the orthogonal ribosome in response to specific environmental signals. The

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degradation circuit is a genetic NOT gate with an output of I-SceI homing

19

endonuclease, which conditionally degrades the orthogonal ribosome genes. We

20

demonstrate that the activation circuit can be flexibly incorporated into genetic

21

circuits and metabolic pathways for encryption. The plasmid-based encryption of the

22

deoxychromoviridans pathway and the genome-based encryption of lacZ are tightly

23

regulated and can decrease the expression to 7.3% and 7.8%, respectively. We

24

validated the ability of the degradation circuit to decrease the expression levels of the

25

target plasmids and the orthogonal rRNA (O-rRNA) plasmids to 0.8% in lab medium

26

and 0.1% in nonsterile soil medium, respectively. Our orthogonal ribosome

27

bio-firewall is a versatile platform that can be useful in biosafety research and in the

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biotechnology industry.

ACS Paragon Plus Environment


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Keywords:

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Bio-firewall, orthogonal ribosome, genetically modified organisms, biosafety, biocontainment,

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synthetic biology

33 34

With the development of biotechnology, artificial biological systems have been

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created for various purposes, such as the production of biochemicals1, bioenergy2, and

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therapeutics3. Moreover, in the past few years, significant progress has been achieved

37

in genome editing and genome synthesis. Hundreds of genomic mutants have been

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successfully incorporated into single molecules of genomic DNA by MAGE4,5 and

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CRISPR6–8. Millions of base-pairs have been successfully assembled in the chemical

40

synthesis of DNA oligos9–11 in genome synthesis. With these advanced technologies,

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artificial biological systems12 with increasing numbers of genes can be designed and

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constructed. Thus far, most of these artificial biosystems for controlling gene

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expression were constructed using regulation elements that were directly compatible

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with the host. However, the complete compatibility of regulation systems between

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artificial biosystems and their host can cause many problems. First, the compatible

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gene regulation system makes the artificial biosystems so transparent to the host that

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the efficiency of regulation is hindered. From a biosafety perspective, there is

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significant potential for uncontrolled leakage of the artificial biosystems into nature.

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Therefore, it is a challenge to design and construct artificial biosystems with high

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biosafety on a large scale.

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Many biocontainment technologies have recently been developed to prevent the

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release of genetically modified organisms (GMOs) into the environment. Gallagher et

53

al.13 developed engineered riboregulators and nucleases to restrict the viability of E.

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coli cells to media containing exogenously supplied synthetic small molecules. Cai et

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al.14 used both transcriptional and recombinational “safeguards” to control essential

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gene functions of Saccharomyces cerevisiae using estradiol. Mandell et al.15


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reconstructed essential enzymes to induce dependence on unnatural amino acids for

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survival. Clement et al.16 designed ‘Deadman’ and ‘Passcode’ switches with different

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signal inputs and killing mechanisms to kill bacteria efficiently. These existing

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biocontainment methods mostly focus on preventing the unintended proliferation of

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genetically modified microorganisms. However, it has been reported that the

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organisms can survive the killing mechanism by spontaneous mutagenesis14,15 or

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horizontal gene transfer17, presumably causing biocontainment problems in an open

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environment or ecosystem.

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Synthetic orthogonal systems are uncoupled from cellular regulation, which makes

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their biology more amenable to engineering. The orthogonal ribosome (O-ribosome)

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specifically translates the orthogonal mRNA (O-mRNA) without cross-talk with the

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endogenous ribosomes and mRNA. Rackham and Chin18 developed methods for the

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selection and characterization of O-ribosome:O-mRNA pairs. Chubiz and Rao19

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developed a computational method for the rational design of O-ribosomes in bacteria.

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An and Chin20 created an orthogonal gene expression pathway in Escherichia coli by

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combining orthogonal transcription by T7 RNA polymerase and translation by an

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O-ribosome. Orelle et al.21 created the first fully orthogonal ribosome–messenger

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RNA system by engineering a hybrid 16S-23S rRNA. O-ribosome systems could be

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developed to perform novel functions without interfering with native translation,

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theoretically enabling the construction of a parallel and independent system.

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Currently, the most common approach to information safety is the password firewall

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system, which utilizes a unique code for authentication to access a resource. To obtain

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access to information from a computer, a specific password is needed to activate the

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system. After one session, the system should log out the user or shut down, which

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deletes the password and silences the system (Fig 1a).

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Here, we have developed a biocontainment system called the O-ribosome bio-firewall,

83

which consists of an activation circuit and a degradation circuit (Fig 1b). The

84

activation circuit is an AND gate22,23, which uses O-ribosomes for specific activation



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of the encrypted pathway. To optimize the orthogonal system, we quantified the

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interaction between the level of orthogonal rRNA (O-rRNA) and the expression levels

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of the target genes. We then developed methods for plasmid-based encryption and

88

genome-based encryption by switching the ribosome binding sites (RBSs) of the

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coding sequences (CDSs) to O-ribosome binding sites (O-RBSs). To prevent the

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horizontal gene transfer of the O-rRNA plasmids, we designed a degradation circuit

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under the control of environmental signals to cleave O-rRNA plasmids. The

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degradation circuit is induced to remove the O-rRNA plasmids in the absence of a

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specific signal, for example, IPTG. Finally, we integrated the activation circuit and the

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degradation circuit together and implemented the bio-firewall in laboratory conditions

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and simulated environmental conditions.

96 97

■ RESULTS AND DISCUSSION

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Design of the activation circuit. To develop a new regulatory system independent

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from the natural expression system, we designed a genetic AND gate23 based on the

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O-ribosome18–20,24,25. Complementary base pair interactions occur between the

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Shine-Dalgarno (SD) sequence of mRNA and the anti-Shine-Dalgarno (ASD)

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sequence of rRNA, guiding the 30S ribosomal subunit to the correct position to

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initiate translation (Fig S1). Orthogonal Shine-Dalgarno (OSD) sequences are mutant

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sequences that are not recognized by the host ribosomes. The mRNAs with OSD

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sequences can be specifically translated by O-ribosomes with mutated ASD sequences

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that are complementary to the OSD sequence. Therefore, it is necessary to

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simultaneously input both the O-mRNA and the O-rRNA to express the target genes

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(Fig 2a). The AND gate was constructed with pBAD and pLac promoters as the two

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inputs to drive the transcription of O-rRNA and O-mRNA, respectively. The

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performance of the AND gate was analyzed under conditions combining different

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concentrations of arabinose and IPTG. When both 1 mM IPTG and 10 mM arabinose

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were present, the O-mRNA and O-rRNA were produced, resulting in maximal red


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fluorescent protein (RFP) fluorescence. When only IPTG or arabinose was present,

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both the pLac and pBAD promoters in the AND gate showed a leakage response. This

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result showed that the activation circuit basically performed the function of an AND

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gate in Boolean logic.

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Characterization of the activation circuit. To characterize our O-ribosome system,

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we investigated the effect of the O-rRNA on the encrypted pathway by quantitative

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real-time PCR (qRT-PCR). Because the 3' end of the 16S rRNA is too short to allow a

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PCR tag primer for detecting the orthogonal 16S rRNA (O-16S rRNA) specifically

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(Fig S2), we designed a special loop at nucleotides 81–89 to identify the O-16S rRNA

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and the host 16S rRNA (Fig 2c). Using the NUPACK software26, we computationally

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designed a new DNA loop with a similar secondary structure to the wild-type (WT)

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16S rRNA25. Meanwhile, the free energy of the secondary structure was also close to

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that of the WT loop (Fig 2c). Nucleotides 81–89 of the WT O-16S rRNA and the

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modified sequence are AGCUUGCUU and GUGAACACU, respectively. The

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secondary structure free energy values of the WT loop and modified loop were -7.30

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kcal/mol and -6.60 kcal/mol, respectively. Orthogonal PCR experiments (Fig S3)

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illustrated that these PCR tags were able to distinguish O-16S rRNA from WT-16S

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rRNA. To investigate the effect of the PCR tag on the activity of the O-ribosomes, the

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expression level of RFP was measured for the two distinct O-16S rRNAs (Fig 3d).

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When IPTG and arabinose were both present, the fluorescence/OD values for the WT

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O-rRNA and modified O-rRNA were 1568 and 1608, respectively. When no inducer

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(IPTG or arabinose) was present, the fluorescence/OD values for the WT O-rRNA and

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modified O-rRNA were 48 and 45, respectively. These results demonstrated that the

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modified O-rRNA preserved the function and activity of WT O-rRNA. This artificial

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loop can be used to track the O-rRNA specifically without interfering with the normal

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rRNA function. Using specific PCR tags, we quantitatively investigated the

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interaction between the expression levels of O-rRNA and of the orthogonal genes. As

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shown in Fig 2e and 2f, the expression levels of O-RFP and O-rRNA increased with



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increasing arabinose concentration. In addition, the expression of RFP was strongly

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associated with the level of O-rRNA. These results demonstrated that high levels of

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O-rRNA improved the expression of O-RFP. According to these results, the high-copy

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plasmid pSB1A3 (~100 copies per cell) was used to enhance the expression of

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O-rRNA and RFP.

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Two noteworthy points are the potential leakage and cell toxicity. First, we analyzed

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the leakiness of O-RBS and O-rRNA, respectively. As shown in Fig S4, the

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fluorescence/OD absorbance values of EJB000, EJB001 and EJB010 are 17, 24 and

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41, respectively. These results show that the O-RBS used in this study has very little

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interaction with the WT-rRNA, and the pBAD promoter accordingly exhibits very

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little leakage expression of O-rRNA in Luria–Bertani broth (LB) medium. After

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background removal, the leakage expression of the orthogonal system accounted for

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1.6% of the maximum expression. Second, the overexpression of O-rRNA is

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potentially toxic to cells due to competition with native rRNA for free ribosomes or to

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translation initiation at unwanted locations on native mRNAs27. We measured the cell

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growth of strains containing WT-rRNA, O-rRNA and empty vector (pSB1A3). As

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shown in Fig S5, cells containing either O-rRNA or WT-rRNA exhibited tiny growth

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defects compared with cells containing empty vector. However, we did not observe

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any growth defect when expressing O-rRNA compared with expressing WT-rRNA.

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These results demonstrated that the orthogonal ribosome system used throughout this

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study had very little effect on cellular viability.

162 163

Encryption for activation circuit. As a proof-of-concept for the activation circuit,

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plasmid-based encryption and decryption were applied to the deoxychromoviridans

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pathway28–30.

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L-tryptophan, catalyzed sequentially by vioA, vioB and vioE. All three genes were

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placed in a polycistron under the control of the pLac promoter (Fig 3a). The medium

Deoxychromoviridans

is

synthesized


heterogeneously

from

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was turned dark green by the expression of VioA-VioB-VioE (pJBVioABE) (Fig 3c).

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To encrypt this synthetic pathway, the SD sequences of vioA, vioB and vioE were

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replaced simultaneously with O-SD sequences (pJBOVioABE). The expression of the

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vioABE genes caused the cells to turn dark green. Tight repression of the vioABE

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gene causes the cells to remain colorless in LB media. The absorbance values of

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EJB020 (Table S1), EJB020 + IPTG, EJB030 (Table S1) and EJB030 + IPTG + Ara

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were 0.117, 0.156, 0.011 and 0.095, respectively. Without IPTG input, the leaky

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expression of the WT vioABE gene turned the cells dark green, with 74.8% of the

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maximum absorbance observed (Fig 3c). No pigments were observed in LB media for

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the strains containing encrypted VioABE. However, the medium turned dark when the

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O-rRNA plasmids were transformed into cells containing the target pathway plasmid

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supplemented with IPTG and Ara. The encryption pathway tightly regulated the

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expression of vioABE and thus decreased the pigment production to 7.3% of that of

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the WT vioABE pathway (Fig 3c). The results illustrated that the O-ribosome system

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provides strict control of the synthetic pathway, which will provide an efficient way to

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control the phenotypes of the encrypted synthetic pathway, even when transformed

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into other bacteria.

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To create the activation circuit to control the pathway on the host genome, a genomic

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encryption method was developed through oligo-mediated replacement31–33, which

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has been proven to be effective in multiplex automated genome engineering4,31,34,35.

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Specifically, the lacZ gene was encrypted as an example (Fig 4a). The original RBS

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sequence and the encrypted RBS sequence before the start codon of the host lacZ are

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CACAGGAAACAGC and TTGTTCCGTCCTCC, respectively. As shown in Fig 4b,

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the WT RBS sequence can be shifted to the O-RBS sequence precisely according to

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the design. The phenotypes of genomic lacZ alleles were analyzed by serial 10-fold

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dilution on LB-X-gal agar plates (Fig 4c). The expression of the lacZ gene turned

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cells blue on the X-gal agar plates. In contrast, cells with the encrypted lacZ gene

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remained white on X-gal agar plates containing IPTG but turned blue upon the



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incorporation of O-rRNA plasmids. Fig 4c shows a quantitative assessment of the

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relative lacZ. The absolute numbers for the absorbance values of EJB041 (Table S1),

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EJB041 + IPTG, EJB060 and EJB060 + IPTG + Ara were 0.372, 0.491, 0.038 and

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0.258, respectively. For the WT RBS, the leakage expression of WT lacZ was 75.7%

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of that of the positive control with IPTG input. For the O-RBS, the background

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expression of encrypted lacZ was only 7.8% of the WT lacZ. Therefore, our activation

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circuit exhibited stricter control of genes expression. The high leakiness of the pLac

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promoter is probably attributable to the WT pLac with a CAP binding site used in this

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study. CAP binding activates transcription in response to the generation of cAMP in a

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low glucose environment36, such as LB medium.

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This biocontainment system integrated O-ribosomes and O-mRNAs to generate the

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output response of genetically modified phenotypes in an encryption-decryption

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process. In plasmid-based encryption, the orthogonal genes of the pathway could be

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quickly reassembled by the Gibson method or yeast assembly method. In the

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genome-based encryption, O-RBSs could be explicitly incorporated into the genome

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by oligo-media replacement or the CRISPR-Cas9 System5,37. This encryption

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approach could theoretically be used for biocontainment control on a larger scale.

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Design of the degradation circuit. To avoid the horizontal transfer of O-rRNA

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plasmids, we designed a genetic NOT gate to cleave O-rRNA plasmids automatically

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in response to environmental signals. This NOT gate was based on the LacI/pTac

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switch and CI/pR switch module consisting of lambda gene cI and its regulatory pR

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promoter. To characterize the NOT gate performance, RFP was first used to profile

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the output of the pR promoter in this NOT gate. Fig 5b shows that the pR promoter

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was significantly inhibited by the repressive effect of IPTG (> 0.1 mM) on CI and

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significantly inducible when IPTG was withdrawn. To digest O-rRNA plasmids

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exclusively, the I-SceI homing endonuclease38,39 was applied. This endonuclease

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specifically recognizes the 18-bp sequence TAGGGATAACAGGGTAAT, which


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does not occur in the E. coli genome and thus in theory should do no harm to the host

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cell. To implement the degradation circuit, RFP was replaced by the I-SceI homing

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endonuclease in the NOT gate (Fig 5c). To optimize the degradation of O-rRNA, two

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versions of the pSB1A3 vector with 1 X I-SceI site and 2 X I-SceI sites were tested.

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The degradation circuit was first integrated into the genome of MG1655Z1. The

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cleavage on the vector with two I-SceI sites was more efficient than that on the vector

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with one I-SceI site (Fig 5c). Therefore, the pSB1A3 vector with two I-SceI sites was

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used as the O-rRNA vector. In addition, to quantify the dynamics of O-rRNA plasmid

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stability with the degradation circuits in the OFF-state (+ IPTG) and the ON-state (-

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IPTG), the fraction of host cells retaining the target plasmid (pSB1A3 with 2 X I-SceI

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sites) was calculated from the ratio of bacteria with red fluorescent emission and the

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total number of bacteria observed. As shown in Fig 5d, the target plasmid maintained

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high stability when IPTG was added to inactivate this NOT ate. In contrast, the target

237

plasmid expression decreased to 0.8% after 36 h of degradation (Fig 5e). These

238

experiments suggested that the target vectors exhibited high genetic stability in the

239

presence of IPTG but were dramatically degraded upon the withdrawal of IPTG. This

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degradation circuit generated NOT gate logic behavior by the control of I-SceI

241

homing endonuclease expression, which could be used to prevent the horizontal gene

242

transfer of target plasmids.

243 244

Integration of the activation circuit and the degradation circuit.

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To prove the concept of the bio-firewall with a designed function, the activation

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circuit and the degradation circuit were integrated in E. coli. The degradation circuit

247

was previously incorporated into the genome of the host cells to obtain the strain

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EJB080. The encrypted deoxychromoviridans pathway and the O-rRNA plasmids

249

with 2 X SceI sites were simultaneously transformed into EJB080 (Fig 6a). To

250

simulate an open environment, we used nonsterile soil medium and examined the

251

microbes present before culturing the bio-firewall strains (Fig S6). Microbe colonies



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of different colors and sizes on LB agar and LB-Amp agar indicated the presence of

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ampicillin-resistant microbes in the soil, while those microbes were sensitive to

254

kanamycin and chloramphenicol. Subsequently, the performance of bio-firewall

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strains in the laboratory environment and in a simulated open environment (nonsterile

256

soil medium) were investigated separately. As shown in Fig 6b and 6c, almost all the

257

colonies produced dark pigments in LB media with arabinose and IPTG, while no

258

dark pigments were observed in nonsterile soil medium. PCR analysis of those

259

colonies showed that the O-rRNA plasmids were stable in the laboratory environment,

260

indicating that the degradation circuit was regulated tightly in the presence of IPTG.

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PCR analysis of those colonies showed that the O-rRNA plasmids were degraded in

262

the simulated open environment, implying that there was insufficient environmental

263

IPTG or lactose to repress the degradation circuit, and thus the I-SceI endonuclease

264

digested the O-rRNA plasmids. In general, even though lactose and arabinose are

265

readily found in the environment, most microbes can use them as carbon sources for

266

cell growth, which reduces their concentrations below the essential inducible

267

concentration for the circuits. Therefore, it is reasonable to believe that the

268

degradation circuit would functionally cleave the O-rRNA plasmids in the open

269

environment.

270

The O-rRNA plasmid might still escape to the environment without plasmid cleavage,

271

when some cells are lysed or when endonuclease expression is deactivated by random

272

mutation. Therefore, quantitative PCR was used to examine the presence of O-rRNA

273

plasmids in the cell culture over long periods of cell cultivation. Fig S7 shows the

274

changes in relative copy number of the target plasmids when the bio-firewall strains

275

were cultured in LB-IPTG-Ara medium and nonsterile soil medium. The O-rRNA

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plasmids in the nonsterile soil medium decreased to nearly 10-2 compared with those

277

in the laboratory medium in 48 h. The O-rRNA plasmids remained stable during

278

long-term cultivation when the degradation circuit was tightly repressed by IPTG but

279

were dramatically diminished in the nonsterile soil medium. There are several reasons


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for this difference. First, cells escaping to the environment grow slower than cells in

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the laboratory medium due to poor nutrition, leading to smaller amounts of O-rRNA

282

plasmid in the soil medium than in the laboratory medium. Second, most O-rRNA

283

plasmids in the nonsterile soil medium were digested by the I-SceI endonuclease

284

expressed in the absence of sufficient environmental IPTG or lactose. Last, the

285

escaping O-rRNA plasmids might be degraded by DNases or microbes (Fig S6) in the

286

soil40.

287 288

The O-ribosome bio-firewall, as described here, represents the state of the art for

289

microbial biocontainment and offers certain advantages. First, using the central role of

290

Watson-Crick base pairing, we can readily modify the bio-firewall by computational

291

modeling and experimental characterization. It is reasonable to choose different

292

O-SD:O-ASD pairs for different target pathways, which will improve the diversity

293

and security of the bio-firewall. Second, due to the small size of RBSs, the O-RBS

294

could be incorporated into genetic circuits and metabolic pathways, enabling flexible

295

engineering. Third, the O-RBS and O-rRNA in this work provide a restrictive control,

296

enabling the efficient construction of complex synthetic biological systems. Here, the

297

pathway-encryption method tightly regulated expression of vioABE, decreasing the

298

pigment production to 7.3% of the level in the WT vioABE pathway, and the

299

expression of encrypted lacZ was decreased to 7.8% of the WT lacZ level. Fourth, our

300

system was more stable because the degradation circuit conditionally digested the

301

O-rRNA plasmid, permanently silencing the expression of the target pathway.

302

Compared with other killing switches13–15, the O-ribosome bio-firewall caused no

303

harm to the host, potentially reducing the risks of unintended proliferation enabled by

304

spontaneous mutagenesis. Finally, in principle, the regulation of those genetic circuits

305

can be extended to respond to different signals due to the modularity of the design.

306

For example, the aTc-induced pTet promoter could replace the pBAD or pTac

307

promoter of our circuits. Our system could be improved to conditionally degrade the



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308

plasmid containing the encrypted pathway by introducing two of the I-SceI sites into

309

this plasmid backbone.

310

Overall, the O-ribosome bio-firewall is a highly efficient, flexible and cost-effective

311

platform for biocontainment. We anticipate the widespread use of the O-ribosome

312

bio-firewall for ecosystem safety in the biotechnology industry.

313 314

■

315

METHODS AND MATERIALS

316

Strains and media. Plasmid cloning work and circuit construct characterization were

317

all performed in the Escherichia coli strain DH10B {F−mcrA ∆(mrr-hsdRMS-mcrBC)

318

Φ80lacZ∆M15 ∆lacX74 recA1 endA1 ara∆139 ∆ara, leu) 7697 galU galK λ-rpsL

319

(StrR) nupG}. MG1655Z1 (F-, λ-, SpR, lacR, tetR), a gift from Michael B Elowitz41.

320

Cells were cultured in Luria–Bertani broth (LB) media (10 g/l peptone, 5 g/l NaCl, 5

321

g/l yeast extract). The nonsterile soil medium consisted of 100 g of fresh garden soil

322

and 900 ml of fresh lake water (Tianjin, postcode 300072). Kanamycin (Kan, 50

323

mg/ml), ampicillin (Amp, 100 mg/ml) and chloramphenicol (Cm, 34 mg/ml) were

324

added as appropriate. Two inducers were obtained from Sigma Aldrich: arabinose

325

(Ara) and isopropyl b-D-1-thiogalactopyranoside (IPTG).

326

Plasmid circuit construction. All plasmids were constructed using standard

327

molecular cloning techniques and Gibson assembly. All plasmid maps are shown in

328

the Supporting Information. Restriction endonucleases, T4 DNA Ligase and Phusion

329

PCR kits from New England BioLabs (NEB) were used. PCR was performed with an

330

ABI Thermal Cycler. Primers were synthesized by Genewiz. All plasmids were

331

transformed into E. coli strain DH10B with standard protocols and isolated with

332

TIANprep Mini Plasmid Kits. Plasmid constructs were verified by restriction digests

333

and sequencing by Genewiz. Details are presented in Supporting Information S4-12,

334

with plasmid maps describing representative circuit constructs. The plasmids pSB1A3,

335

pSB3C5 and pSB4K5 were from the Registry of Standard Biological Parts


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336

(http://partsregistry.org). The yeast/E. coli shuttle vector pJB413C was constructed

337

using multichange isothermal mutagenesis by replacing the beta lactamase (bla) gene

338

and Ori site of pRS413 with the CmR gene and p15A ori (Supporting Information Fig

339

S6). Specifically, pRS413 was amplified by PCR with primer pairs that produce two

340

PCR products with homologous ends. After gel purification, the two DNA fragments

341

were assembled by Gibson assembly42.

342

Pathway encryption in plasmid. The pJB413C plasmid was linearized by SacI and

343

BamHI. The WT pJBVioABE (Supporting Information Fig S7) was assembled by

344

transforming linear pJB413C and the PCR products of SD-VioA, SD-VioB, and

345

SD-VioC into yeast. The encryption pathway of pJBOVioABE was assembled by

346

transforming linear pJB413C and the PCR products of OSD-VioA, OSD-VioB, and

347

OSD-VioC into yeast. Yeast transformants were combined into a single pool for

348

plasmid recovery into E. coli DH10B, where blue/white screening distinguished

349

empty vector from clones in which the inserts were assembled. Colony PCR was used

350

to identify clones encoding full-length inserts to be sent for sequencing. (See

351

RADOM43).

352

Pathway encryption in genome. Strain MG1655Z1 containing pKD4644 was grown

353

at 32°C to midlog (OD600 = 0.4−0.6). λ-Red was induced with the addition of 10 mM

354

L-arabinose and incubated for 1 h, then quick-chilled in ice for 10 min. To prepare for

355

electroporation, 1 ml of cells was washed twice with cold sterile ddH2O and finally

356

concentrated 20-fold in 50 µl of cold sterile ddH2O. To modify the genome via

357

electroporation, 1 µM oligo 5'-GTATGTTGTGTGGAATTGTGAGCGGATAACA

358

ATTTCATTGTTCCGTCCTCCATGACCATGATTACGGATTCACTGGCCGTCG

359

TTTTACA-3' was mixed into 50 µl electrocompetent cells and placed in a Bio-Rad

360

electroporator (0.1 cm cuvette, 1.80 kV). After electroporation, cells were allowed to

361

recover for 3 h in LB before plating for blue/white screening. β-Galactosidase activity

362

was measured using the Yeast β-Galactosidase Assay Kit (Thermo Scientific).

363

Relative fluorescence measurements. Single colonies were used to inoculate LB (5



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Page 14 of 29

364

ml) overnight in a shaking incubator at 37 ℃ and 220 rpm. Cultures were

365

back-diluted to OD600 0.5–0.6 into LB (5 ml) containing appropriate antibiotics and

366

inducers. The cultures were then grown in a shaking incubator for 8 h at 37°C and 220

367

rpm. Then, 200 µL cell culture was transferred to a 96-well fluorescence plate, and the

368

optical density was read at 600 nm. RFP fluorescence measurements were performed

369

on a Spectramax M2 (Molecular Devices) with excitation at 584 nm and measurement

370

of the emission at 617 nm. The experiment was performed in triplicate. The

371

Fluorescence/OD was the fluorescence per OD600. The Relative Fluorescence/OD is

372

reported as the fraction of the maximum Fluorescence/OD.

373

Quantitative real-time PCR. To measure the performance of the O-ribosomes at the

374

RNA level, the relative rRNA concentrations were measured with quantitative PCR.

375

We use the relative gene expression (the 2-∆∆Ct method45) to present the data of the

376

O-16S rRNA relative to the internal control gene the WT-23S rRNA. The growth

377

conditions for the collection of total RNA were as described above. Total RNA was

378

extracted with the RNeasy Protect Bacteria Mini Kit (Qiagen), according to the

379

manufacturer’s instructions. First-strand cDNA was synthesized with the SuperScript

380

III First-Strand Synthesis System for RT-PCR (Invitrogen). Random hexamers were

381

used for reverse transcription. Fast SYBR Green Master Mix (Applied Biosystems)

382

was used for real-time PCR, and experiments were performed on the StepOnePlus

383

Real-Time PCR System (Applied Biosystems). The target primers of O-16S rRNA

384

were

385

(5'-GCCTAGGTGAGCCGTTACCC-3'), and the reference primers of WT-23S rRNA

386

were

387

(5'-CCTCGGGGTACTTAGATGTT-3'). The relative O-rRNA lever is reported as the

388

fold changes of the background O-rRNA lever.

389

Fluorescent-based plasmids degradation assay. The degradation circuit was

390

linearized by ApaI and integrated into the genome of MG1655Z1 by λ-red

391

recombination44 (EJB080). Single colonies of EJB080 were used to inoculate LB (1

P1

P3

(5'-TAACAGGAAGAGTGAACACT-3')

(5'-CTAAGCGTACACGGTGGATG-3')


and

and

P2

P4

Page 15 of 29

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392

ml) containing kanamycin and 1 mM IPTG, and the resulting liquid cultures were

393

grown in a shaking incubator for 3 h at 37 ℃ and 250 rpm. until OD600 0.5–0.6. This

394

culture was used to prepare electrocompetent cells. Two of the RFP target plasmids

395

(pJB1SceI and pJB2SceI) were transformed into electrocompetent cells (EJB080),

396

plated onto LB agar (ampicillin, kanamycin and 1 mM IPTG) and grown for 12 h at

397

37°C. Single colonies (EJB090 and EJB100) were used to inoculate 1 ml of LB

398

(ampicillin, kanamycin and 1 mM IPTG) and grown in a shaking incubator at 37°C

399

until OD600 0.5–0.6. The cultures were spun down at 12,000 g, washed once with

400

fresh LB (1 ml) and back-diluted to OD600 0.01 in 20 ml of LB containing 1 mM

401

IPTG for the OFF-state and 0 mM IPTG for ON-state, respectively. RFP fluorescence

402

imaging and phage imaging were then obtained from this cell lawn using a

403

fluorescence microscope (Olympus CX41). The fraction of host cells retaining the

404

target plasmid (Target+) was calculated from the ratio of bacteria exhibiting red

405

fluorescent emission to the total bacteria numbers observed.

406

O-rRNA plasmid degradation assay

407

The O-rRNA plasmids (pJB2SORibo2) and pJBOVioABE were transformed into

408

electrocompetent cells (EJB080), plated onto LB agar (ampicillin, kanamycin,

409

chloramphenicol and 1 mM IPTG) and grown for 12 h at 37°C. In the laboratory

410

environment, single colonies (EJB110) were used to inoculate 20 ml of LB medium

411

(ampicillin, kanamycin, chloramphenicol and 1 mM IPTG) and grown in a shaking

412

incubator at 37℃. Cultures were diluted appropriately after 48 h, plated onto LB agar

413

(ampicillin, kanamycin, chloramphenicol and 1 mM IPTG) and grown at 37°C. For

414

the open environment, single colonies (EJB110) were used to inoculate 20 ml of

415

nonsterile soil medium and grown in a shaking incubator at 37°C. Cultures were

416

diluted appropriately after 48 h, plated onto LB agar (kanamycin, chloramphenicol)

417

and grown at 37 ℃. P5 (5'-TGCCACCTGACGTCTAAGAA-3') and P6

418

(5'-TCGCCACCGTCGGTCGCAATG-3') were used for PCR analysis of the O-rRNA



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

419

plasmids. To examine the presence of O-rRNA plasmids in the cell culture over a long

420

period of cell cultivation, 1 ml sterile cultures (12 h, 24 h, 36 h, 48 h, 60 h) from both

421

of the media described above were collected by centrifugation (12000 rpm, 5 min)

422

and then filtrated through a 0.22-µm aqueous membrane, respectively. The O-rRNA

423

plasmids were also detected by PCR analysis (P5 and P6) and electrophoresis on a 1%

424

agarose gel. The primers P7 (5'- GATAACGAGCTCCTGCACTG -3') and P8 (5'-

425

ACTGTGAGCCAGAGTTGCCC -3') were used to target the chromosomal lacZ as

426

reference gene.

427

ASSOCIATED CONTENT

428

Supporting Information

429

Table S1, supplementary figures (Fig S1-S7), plasmid maps (Fig S8-S15). This

430

material is available free of charge via the Internet at http://pubs.acs.org. In addition,

431

The plasmids produced in this work may be obtained from the SynbioML repository

432

by request (http://www.synbioml.org/ModuleLib/index.jsp).

433

AUTHOR INFORMATION

434

Corresponding Author

435

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

436

Author Contributions

437

B.J. was the first author and designed most of the experiments; B.J., S.P., D.L. H.L.

438

and Y-Z.C performed the research; B.J., and Y-Z.C. designed and constructed the

439

genetic circuits, and S.P. and D.L. assembled the vioABE pathways. H.L. detected

440

and analyzed the products of the vioABE pathways. B.J., H.Q., and B-Z.L. wrote the

441

manuscript; Y-Z.C. improved the manuscript; and all the work described was guided

442

by Y-J.Y.


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443

■

444

Notes

445

The authors declare no competing financial interests.

446

■

447

ACKNOWLEDGMENTS

448

This work was funded by the Ministry of Science and Technology of China (“973”

449

Program, 2014CB745100), the International S&T Cooperation Program of China

450

(2015DFA00960), and the National Natural Science Foundation of China (21390203

451

and 21621004). We thank the Tianjin University 2012 iGEM team for their initial

452

work and ideas on the O-Key system. We thank Michael B. Elowitz for providing the

453

MG1655Z1 strains.

454

■

455

ABBREVIATIONS

456

RBS, ribosome binding sites; SD, Shine-Dalgarno; ASD, Anti-Shine-Dalgarno; CDS:

457

coding sequence; PCR, polymerase chain reaction; bp, base pair; kb, kilobase pair;

458

rRNA, ribosomal RNA; O-rRNA, orthogonal ribosomal RNA; WT, wild type; O-,

459

orthogonal-; Kan, kanamycin; Amp, ampicillin; Cm, chloramphenicol.

460

■

461

REFERENCES

462

(1) Smanski, M. J., Zhou, H., Claesen, J., Shen, B., Fischbach, M. A., and Voigt, C. A.

463

(2016) Synthetic biology to access and expand nature’s chemical diversity. Nat. Rev.

464

Microbiol. 14, 135–149.

465

(2) Atsumi, S., Hanai, T., and Liao, J. C. (2008) Non-fermentative pathways for

466

synthesis of branched-chain higher alcohols as biofuels. Nature 451, 86–9.

467

(3) Lienert, F., Lohmueller, J. J., Garg, A., and Silver, P. a. (2014) Synthetic biology in

468

mammalian cells: next generation research tools and therapeutics. Nat. Rev. Mol. Cell

469

Biol. 15, 95–107.



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

470

(4) Wang, H. H., Kim, H., Cong, L., Jeong, J., Bang, D., and Church, G. M. (2012)

471

Genome-scale promoter engineering by coselection MAGE. Nat Methods 9, 591–593.

472

(5) Ronda, C., Pedersen, L. E., Sommer, M. O. A., and Nielsen, A. T. (2016) CRMAGE:

473

CRISPR Optimized MAGE Recombineering. Sci. Rep. 6, 19452.

474

(6) Konermann, S., Brigham, M. D., Trevino, A. E., Joung, J., Abudayyeh, O. O.,

475

Barcena, C., Hsu, P. D., Habib, N., Gootenberg, J. S., Nishimasu, H., Nureki, O., and

476

Zhang, F. (2014) Genome-scale transcriptional activation by an engineered

477

CRISPR-Cas9 complex. Nature 2–11.

478

(7) DiCarlo, J. E., Chavez, A., Dietz, S. L., Esvelt, K. M., and Church, G. M. (2015)

479

Safeguarding CRISPR-Cas9 gene drives in yeast. Nat. Biotechnol. 33, 1250–1255.

480

(8) Caliando, B. J., and Voigt, C. A. (2015) Targeted DNA degradation using a

481

CRISPR device stably carried in the host genome. Nat. Commun. 6, 6989.

482

(9) Ostrov, N., Landon, M., Guell, M., Kuznetsov, G., Teramoto, J., Cervantes, N.,

483

Zhou, M., Singh, K., Napolitano, M. G., Moosburner, M., Shrock, E., Pruitt, B. W.,

484

Conway, N., Goodman, D. B., Gardner, C. L., Tyree, G., Gonzales, A., Wanner, B. L.,

485

Norville, J. E., Lajoie, M. J., and Church, G. M. (2016) Design, synthesis, and testing

486

toward a 57-codon genome. Science 353, 819–822.

487

(10) Annaluru, N., Muller, H., and Mitchell, L. (2014) Total Synthesis of a Functional

488

Designer Eukaryotic Chromosome. Science 344, 55–58.

489

(11) Dymond, J. S., Richardson, S. M., Coombes, C. E., Babatz, T., Muller, H.,

490

Annaluru, N., Blake, W. J., Schwerzmann, J. W., Dai, J., Lindstrom, D. L., Boeke, A.

491

C., Gottschling, D. E., Chandrasegaran, S., Bader, J. S., and Boeke, J. D. (2011)

492

Synthetic chromosome arms function in yeast and generate phenotypic diversity by

493

design. Nature 477, 471–476.

494

(12) Nandagopal, N., and Elowitz, M. B. (2011) Synthetic biology: integrated gene

495

circuits. Science 333, 1244–8.


Page 18 of 29

Page 19 of 29

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


496

(13) Gallagher, R. R., Patel, J. R., Interiano, A. L., Rovner, A. J., and Isaacs, F. J. (2015)

497

Multilayered genetic safeguards limit growth of microorganisms to defined

498

environments. Nucleic Acids Res. 43, 1945–54.

499

(14) Cai, Y., Agmon, N., Choi, W. J., Ubide, A., Stracquadanio, G., Caravelli, K., Hao,

500

H., Bader, J. S., and Boeke, J. D. (2015) Intrinsic biocontainment: multiplex genome

501

safeguards combine transcriptional and recombinational control of essential yeast

502

genes. Proc. Natl. Acad. Sci. U. S. A. 112, 1803–8.

503

(15) Mandell, D. J., Lajoie, M. J., Mee, M. T., Takeuchi, R., Kuznetsov, G., Norville, J.

504

E., Gregg, C. J., Stoddard, B. L., and Church, G. M. (2015) Biocontainment of

505

genetically modified organisms by synthetic protein design. Nature 518, 55–60.

506

(16) Chan, C. T. Y., Lee, J. W., Cameron, D. E., Bashor, C. J., and Collins, J. J. (2015)

507

“Deadman” and “Passcode” microbial kill switches for bacterial containment. Nat.

508

Chem. Biol. 1–7.

509

(17) Wright, O., Delmans, M., Stan, G. B., and Ellis, T. (2015) GeneGuard: A modular

510

plasmid system designed for biosafety. ACS Synth. Biol. 4, 307–316.

511

(18) Rackham, O., and Chin, J. W. (2005) A network of orthogonal ribosome x mRNA

512

pairs. Nat Chem Biol 1, 159–166.

513

(19) Chubiz, L. M., and Rao, C. V. (2008) Computational design of orthogonal

514

ribosomes. Nucleic Acids Res 36, 4038–4046.

515

(20) An, W., and Chin, J. W. (2009) Synthesis of orthogonal transcription-translation

516

networks. Proc. Natl. Acad. Sci. U. S. A. 106, 8477–8482.

517

(21) Orelle, C., Carlson, E. D., Szal, T., Florin, T., Jewett, M. C., and Mankin, A. S.

518

(2015) Protein synthesis by ribosomes with tethered subunits. Nature.

519

(22) Wang, B., Kitney, R. I., Joly, N., and Buck, M. (2011) Engineering modular and

520

orthogonal genetic logic gates for robust digital-like synthetic biology. Nat. Commun. 2,

521

508.



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

522

(23) Moon, T. S., Lou, C., Tamsir, A., Stanton, B. C., and Voigt, C. A. (2012) Genetic

523

programs constructed from layered logic gates in single cells. Nature 491, 249–253.

524

(24) Rackham, O., and Chin, J. W. (2005) Cellular logic with orthogonal ribosomes. J.

525

Am. Chem. Soc. 127, 17584–5.

526

(25) Barrett, O. P., and Chin, J. W. (2010) Evolved orthogonal ribosome purification

527

for in vitro characterization. Nucleic Acids Res 38, 2682–2691.

528

(26) Zadeh, J. N., Steenberg, C. D., Bois, J. S., Wolfe, B. R., Pierce, M. B., Khan, A. R.,

529

Dirks, R. M., and Pierce, N. A. (2011) NUPACK: Analysis and design of nucleic acid

530

systems. J. Comput. Chem. 32, 170–173.

531

(27) Yang, C., Hockenberry, A. J., Jewett, M. C., and Amaral, L. A. N. (2016)

532

Depletion of Shine-Dalgarno Sequences within Bacterial Coding Regions Is

533

Expression Dependent. G3 (Bethesda). X, 1–9.

534

(28) Torella, J. P., Boehm, C. R., Lienert, F., Chen, J.-H., Way, J. C., and Silver, P. a.

535

(2014) Rapid construction of insulated genetic circuits via synthetic sequence-guided

536

isothermal assembly. Nucleic Acids Res. 42, 681–9.

537

(29) Temme, K., Hill, R., Segall-Shapiro, T. H., Moser, F., and Voigt, C. A. (2012)

538

Modular control of multiple pathways using engineered orthogonal T7 polymerases.

539

Nucleic Acids Res 40, 8773–8781.

540

(30) Lee, M. E., Aswani, A., Han, A. S., Tomlin, C. J., and Dueber, J. E. (2013)

541

Expression-level optimization of a multi-enzyme pathway in the absence of a

542

high-throughput assay. Nucleic Acids Res. 41, 10668–78.

543

(31) Dicarlo, J. E., Conley, A. J., Penttilä, M., Jäntti, J., Wang, H. H., and Church, G. M.

544

(2013) Yeast oligo-mediated genome engineering (YOGE). ACS Synth. Biol. 2, 741–

545

749.

546

(32) Wang, H. H., Xu, G., Vonner, A. J., and Church, G. (2011) Modified bases enable

547

high-efficiency oligonucleotide-mediated allelic replacement via mismatch repair


Page 20 of 29

Page 21 of 29

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


548

evasion. Nucleic Acids Res. 39, 7336–47.

549

(33) Li, Y., Gu, Q., Lin, Z., Wang, Z., Chen, T., and Zhao, X. (2013) Multiplex Iterative

550

Plasmid Engineering for combinatorial optimization of metabolic pathways and

551

diversification of protein coding sequences. ACS Synth. Biol.

552

(34) Isaacs, F. J., Carr, P. A., Wang, H. H., Lajoie, M. J., Sterling, B., Kraal, L.,

553

Tolonen, A. C., Gianoulis, T. A., Goodman, D. B., Reppas, N. B., Emig, C. J., Bang, D.,

554

Hwang, S. J., Jewett, M. C., Jacobson, J. M., and Church, G. M. (2011) Precise

555

manipulation of chromosomes in vivo enables genome-wide codon replacement.

556

Science 333, 348–353.

557

(35) Wang, H. H., Isaacs, F. J., Carr, P. A., Sun, Z. Z., Xu, G., Forest, C. R., and Church,

558

G. M. (2009) Programming cells by multiplex genome engineering and accelerated

559

evolution. Nature 460, 894–898.

560

(36) Yu, X.-M., and Reznikoff, W. S. (1984) Deletion analysis of the CAP-cAMP

561

binding site of the Escherichia coli lactose promoter. Nucleic Acids Res. 12, 5449–

562

5464.

563

(37) Li, Y., Lin, Z., Huang, C., Zhang, Y., Wang, Z., Tang, Y. jie, Chen, T., and Zhao,

564

X. (2015) Metabolic engineering of Escherichia coli using CRISPR-Cas9 meditated

565

genome editing. Metab. Eng. 31, 13–21.

566

(38) Lin, Z., Xu, Z., Li, Y., Wang, Z., Chen, T., and Zhao, X. (2014) Metabolic

567

engineering of Escherichia coli for the production of riboflavin. Microb. Cell Fact. 13,

568

104.

569

(39) Mitchell, L. a., and Boeke, J. D. (2014) Circular permutation of a synthetic

570

eukaryotic chromosome with the telomerator. Proc. Natl. Acad. Sci. U. S. A 111.

571

(40) Nielsen, K. M., Johnsen, P. J., Bensasson, D., and Daffonchio, D. (2007) Release

572

and persistence of extracellular DNA in the environment. Environ. Biosaf. Res 6, 37–

573

53.



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

574

(41) Cox 3rd, R. S., Dunlop, M. J., and Elowitz, M. B. (2010) A synthetic three-color

575

scaffold for monitoring genetic regulation and noise. J Biol Eng 4, 10.

576

(42) Gibson, D. G., Young, L., Chuang, R. Y., Venter, J. C., Hutchison 3rd, C. A., and

577

Smith, H. O. (2009) Enzymatic assembly of DNA molecules up to several hundred

578

kilobases. Nat Methods 6, 343–345.

579

(43) Lin, Q., Jia, B., Mitchell, L. A., Luo, J., Yang, K., Zeller, K. I., Zhang, W., Xu, Z.,

580

Stracquadanio, G., Bader, J. S., and Boeke, J. D. (2014) RADOM, an Efficient In Vivo

581

Method for Assembling Designed DNA Fragments up to 10 kb Long in Saccharomyces

582

cerevisiae. ACS Synth. Biol. 4, 213–20.

583

(44) Datsenko, K. A., and Wanner, B. L. (2000) One-step inactivation of chromosomal

584

genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. U. S. A 97,

585

6640–6645.

586

(45) Schmittgen, T. D., and Livak, K. J. (2008) Analyzing real-time PCR data by the

587

comparative CT method. Nat. Protoc. 3, 1101–1108.

588


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Table of Contents/Abstract Graphic 79x59mm (300 x 300 DPI)



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

Fig 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 bio-firewall system was designed for biocontainment and biosafety. 314x177mm (300 x 300 DPI)


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Fig 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 OmRNA. (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 Oribosomes (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 detail). 348x343mm (300 x 300 DPI)



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Fig 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 extracted29, 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.) 337x172mm (300 x 300 DPI)


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Fig 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.) 344x183mm (300 x 300 DPI)



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Fig 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 co-transformed with the degradation circuits and the target vector with one and two I-SceI sites were re-streaked 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.


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Fig 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. 302x323mm (300 x 300 DPI)


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Orthogonal Ribosome Biofirewall - ACS Publications - American

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