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Developing a Synthetic Biology Toolkit for Comamonas testosteroni, an Emerging Cellular Chassis for Bioremediation Qiang Tang, Ting Lu, and Shuang-Jiang Liu ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.7b00430 • Publication Date (Web): 03 Jun 2018 Downloaded from http://pubs.acs.org on June 5, 2018

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ACS Synthetic Biology

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Developing a Synthetic Biology Toolkit for Comamonas testosteroni,

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an Emerging Cellular Chassis for Bioremediation

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Qiang Tang1,2,3, Ting Lu3-6*, and Shuang-Jiang Liu1,2,7*

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Beijing, 100101, China;

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University of Chinese Academy of Sciences, Beijing 100049, China;

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Department of Bioengineering, 4 Department of Physics, 5 Center for Biophysics and

State Key Laboratory of Microbial Resources, Chinese Academy of Sciences,

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Quantitative Biology,

Carl R. Woese Institute for Genomic Biology, University of

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Illinois at Urbana-Champaign, Urbana, IL 61801, USA;

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Beijing, 100101, China

Environmental Microbiology Research Center, Chinese Academy of Sciences,

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* Corresponding authors:

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Shuang-Jiang Liu (Beichen Xilu 1, Chaoyang district, Beijing 100101, China. Tel:

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+86-10-64807423, Fax: +86-10-64807421, Email: [email protected]) and Ting Lu (1304

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West Springfield Avenue, Urbana, IL 61801, USA. Tel: +1-217-333-4627. Fax: +1-

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217-265-0246. Email: [email protected])

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1 ACS Paragon Plus Environment

ACS Synthetic Biology 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

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Abstract:

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Synthetic biology is rapidly evolving into a new phase that emphasizes real-world

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applications such as environmental remediation. Recently, Comamonas testosteroni

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has become a promising chassis for bioremediation due to its natural pollutant-

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degrading capacity; however, its application is hindered by the lack of fundamental

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gene expression tools. Here, we present a synthetic biology toolkit that enables rapid

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creation of functional gene circuits in C. testosteroni. We first built a shuttle system

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that allows efficient circuit construction in E. coli and necessary phenotypic testing in

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C. testosteroni. Then, we tested a set of wildtype inducible promoters, and further

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used a hybrid strategy to create engineered promoters to expand expression strength

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and dynamics. Additionally, we tested the T7 RNA Polymerase-PT7 promoter system

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and reduced its leaky expression through promoter mutation for gene expression. By

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coupling random library construction with FACS screening, we further developed a

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synthetic T7 promoter library to confer a wider range of expression strength and

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dynamic characteristics. This study provides a set of valuable tools to engineer gene

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circuits in C. testosteroni, facilitating the establishment of the organism as a useful

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microbial chassis for bioremediation purposes.

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Keywords: Synthetic biology toolkit, shuttle vehicle, inducible promoter, T7 RNA

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polymerase-PT7 system, PT7DOM promoter library, Comamonas testosteroni


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Introduction

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Over the past eighteen years, synthetic biology has emerged as an

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interdisciplinary field for cellular functionality programming1-4. In addition to many

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proof-of-concept demonstrations, there has been an increasing number of new

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applications, such as environmental bioremediation. Successful examples include the

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construction of microbial biosensors5, and computer-assisted engineering of synthetic

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degradation pathways6. These efforts mainly focused on circuit programming aspects

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that were mostly implemented in highly adapted laboratory organisms such as

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Escherichia coli. As synthetic biology begins to address important real-world

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problems, model laboratory organisms are no longer adequate because of their lack of

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fitness and robustness in natural settings7. Instead, to enable practical applications, it

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is of primary importance to explore natural, field-robust organisms that can enable

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novel synthetic circuits to operate robustly in fields8.

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Comamonas testosteroni, a Gram-negative rod-shaped bacterium, possesses

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many traits that make it an attractive chassis host for environmental bioremediation.

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First, it is a native colonizer of diverse environments, including soils, activated sludge,

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and fresh and marine sediments9. Second, it does not assimilate sugars and, thus, does

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not compete for carbohydrates with other environmental microbes. Third, they

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assimilate xeno compounds like hormone pollutant steroids10-11 and aromatic

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compounds such as 3, 4-dichloroaniline12, dichloroethenes13 and vanillate14. Fourth, C.

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testosteroni is able to aerobically reduce heavy metals15. Fifth, it naturally senses and

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pursues xenobiotic compounds through chemotaxis14, 16. Sixth, C. testosteroni has a 3 ACS Paragon Plus Environment


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strong adaption to changing environments and xenobiotics17-19. With these attractive

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features, researchers have begun to explore C. testosteroni strains as environmental

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bioremediation hosts20-21. Among various strains, C. testosteroni CNB-1 is

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particularly attractive, owing to the existence of the plant-microbe association

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rhizoremediation system22, the system-level studies of genome and proteomics23-25,

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and the well elucidated genetic and biochemical mechanisms14,26.

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Well-refined genetic tools are critical to translate the engineering of C.

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testosteroni into field applicable technologies. There are several highly valuable

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efforts for genetic manipulations in C. testosteroni14, 16; however, compared to model

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organisms such as E. coli27-28, and industrially important bacteria such as

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Corynebacterium glutamicum29-30, which possess rich genetic arsenals, C. testosteroni

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has very limited tools for genetic manipulation. Indeed, the state-of-art of C.

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testosteroni engineering is in its infancy, which motivated us to develop a useful set

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of genetic tools for C. testosteroni for bioremediation purposes.

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Here, we present a synthetic biology toolkit containing shuttle vehicles and a set

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of well characterized gene expression systems. We first constructed E. coli-C.

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testosteroni shuttle vehicles for building and hosting genetic circuits. Then we

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characterized a number of inducible promoters adapted from E. coli, and improved the

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promoter strength and dynamics range by creating IPTG-inducible promoters through

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a hybrid strategy. T7 RNA polymerase (T7RNAP) and its cognate promoter PT7 were

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adopted to drive efficient gene expression. To reduce system leakage, a promoter

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mutant (PT7DO) was engineered. Additionally, to further expand expression strength 4 ACS Paragon Plus Environment

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and dynamics, a library of orthogonal promoters (PT7DOM) was constructed. This

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synthetic biology toolkit paves the way for rapidly engineering of C. testosteroni, thus

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advancing synthetic biology for applications in environmental bioremediation.

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Results and Discussion

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Constructing an E. coli-C. testosteroni shuttle system

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In order to rapidly engineer and stably host genetic circuits in C. testosteroni, we

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started by creating a novel E. coli-C. testosteroni shuttle system that allows for quick

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circuit assembly in E. coli and stable propagation in C. testosteroni.

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As shown in Figure 1A, the shuttle vehicle consists of multiple cloning sites for

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gene circuit insertion, a selection marker, and two replicons for E. coli and C.

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testosteroni, respectively. The p15A origin allows for constructing and tuning genetic

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circuits in E. coli31, while the replication of the endogenous incP-1β plasmid allows

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for plasmid propagation in C. testosteroni. This design enables rapid genetic circuit

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construction in E. coli through which effective DNA assembly and optimization

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strategies were developed. Meanwhile, it allows quick phenotypic validation in C.

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testosteroni through direct transformation.

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In building the shuttle system, we started by designing and constructing the

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vector pEC01K/T in E. coli (Figure 1B and Supplementary Figure S1). In the design,

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the kanamycin and tetracycline resistance genes are used as the marker for pEC01K

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and pEC01T respectively. The multiple cloning sites of the vectors allow for rapid

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gene circuit insertion. In addition, we used p15A as the origin of replication for the



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plasmid to propagate in E. coli. We also searched for the replication origin for

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plasmid propagation in C. testosteroni, and found that the origin in the endogenous

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91-kb plasmid pCNB1, which belongs to the incP-1β group24, offers the required trait.

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Inside pCNB1, there are the replication initiation protein gene trfA1, located at

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positions 17,479-18,702 bp, and the origin oriV at positions 10995-11301 bp that is

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about 6 kb away from trfA1. To utilize the origin, we eliminated the endogenous

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plasmid pCNB1 in our recent study to generate a plasmid free strain C. testosteroni

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PC32. We also showed that C. testosteroni PC transformed with plasmids containing

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trfA1 or oriV alone (p15Kan-trfA and p15Kan-oriV respectively) failed to form any

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colonies; in contrast, C. testosteroni PC transformed with the plasmid pEC01K that

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contains both trfA1 and oriV was able to form colonies, confirming that the plasmid

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pEC01K stably propagates in C. testosteroni PC and that both trfA1 and oriV are

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needed for proper propagation. We further extracted and sequenced the plasmid from

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C. testosteroni PC to confirm that pEC01K resides in the strain. Lastly, to examine

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the metabolic load of the shuttle system, we measured the growth rates of C.

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testosteroni PC and C. testosteroni harboring pEC01K/T (Supplementary Figure S2),

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showing that the shuttle does not cause any major growth defects to C. testosteroni

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PC.

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The resulting shuttle system was evaluated in terms of plasmid transformation

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efficiency and stability. Our study showed that the transformation efficiency of

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pEC01K was 3.3±0.6 ×108 CFU/µg plasmid DNA. A comparable transformation

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efficiency was detected with the plasmid pEC01T, confirming that the vehicles has a


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high transformation efficiency into C. testosteroni PC. To determine the plasmid

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stability, C. testosteroni PC/pEC01K was cultured in LB medium in the presence and

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absence of kanamycin for about 100 generations. Then, the culture was diluted and

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plated on LB agar plates without kanamycin. Finally, colonies were picked to test

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their antibiotic resistance. Our study showed that the plasmid is 100% stable for 100

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generations.

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Evaluating an inducible promoter set

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Promoters are the key to drive gene transcription and expression. In particular,

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inducible promoter systems are useful because they minimize metabolic burden.

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Therefore, we evaluated a number of inducible promoters for use in conjugation with

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the shuttle vehicle pEC01K. Due to their extensive use in E. coli, we selected araC-

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PBAD33, lacI-PLAC34, lacIQ-PTAC35, and lacIQ-PTRC35 as candidates for characterization

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using a GFPmut3b reporter. To enable rapid assembly and exchange of genetic

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elements, a combination of the Gibson assembly and the conventional recombinant

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DNA technique of restriction digestion were applied (Figure 1C), which facilitate

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reusing of genetic fragments.

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The four promoters were first evaluated with their impacts on the growth of the

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resulting strains, C. testosteroni PC harboring the different versions of the inducible

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promoters (Supplementary Figure S3A). When the IPTG induction was below 5 mM,

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there was no observable effects on growth. However, when the IPTG level reached 10

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mM, a growth repression was observed for the strains with the promoters PLAC, PTAC



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and PTRC, probably due to the toxicity of IPTG at a high concentration. We also found

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that C. testosteroni CNB-1 did not grow in the medium where L-Arabinose was the

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sole carbon source (Supplementary Figure S3B), ruling out the possibility that C.

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testosteroni CNB-1 metabolize L-arabinose. The result is also consistent with the fact

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that there is no metabolic genes associated with L-arabinose utilization in the genome

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of C. testosteroni CNB-1. However, we noticed that some growth repression and

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delay was observed when the L-arabinose level reached 100 mM.

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The promoters were subsequently characterized in terms of their strengths (Fig.

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1D). We found that the PLAC promoter was below the detection limit of the microplate

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reader upon 0 to 10 mM of IPTG induction, and only observed through X-Gal

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staining when the promoter was used to drive lacZ (Supplementary Figure S4). Thus,

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the promoter PLAC is the weakest among the four tested promoters. The promoter

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lacIQ-PTAC spans over a 45-fold dynamic range with its maximum at the 2.5 mM IPTG

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induction. The promoter lacIQ-PTRC spans a 7-fold dynamic range, with its maximum

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at the 1 mM induction. The promoter lacIQ-PTRC also a high basal expression. The

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promoter araC-PBAD spans a 5-fold dynamic range, with the maximum occurred at the

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50-100 mM of induction. Among these three promoters, lacIQ-PTRC has the highest

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leaky expression.

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Redesigning the hybrid IPTG inducible promoters

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Although the above four inducible promoters showed successful gene expression

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upon induction, they were relatively weak. In fact, we did not observe any detectable

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signal for the synthetic promoters in a microplate reader whose gain was set to


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appropriately monitor the expression from the T7 promoter in E. coli. In addition,

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they had relative narrow induction ranges, limiting their utilization in C. testosteroni

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PC. Thus, we proceeded to develop two IPTG-inducible promoters by adopting a

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hybrid strategy36. Specifically, one and two copies of the variant of the LacI binding

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operator variant, Oid36, were added at the downstream of PCN, a constitutive strong

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promoter of C. testosteroni32, to generate hybrid inducible promoters PCNOID and

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PCN2OID, respectively (Figure 2A and Supplementary Figure S5). In addition, the intact

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lacI gene was added at the upstream of hybrid promoters in the opposite direction. To

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examine the effects of IPTG induction on cellular growth, we conducted growth

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experiments for the strain C. testosteroni PC/pEC01K-PCNOID under different levels of

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IPTG induction (Figure S6). The result showed that there was no effect on growth for

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IPTG concentrations up to 5 mM, and an observable inhibition to growth at 10 mM

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due to the toxicity of a high IPTG concentration.

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The resulting hybrid promoters showed a high degree of induction sensitivity and

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a large dynamic range. As shown in Figure 2B, the promoter PCNOID responded to

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IPTG induction at a concentration as low as 5 µM, and had a 51 fold dynamic range

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with its maximum induction at 0.5 mM. The promoter PCN2OID, responded to IPTG at

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a level as low as 10 µM, and spanned over a 32-fold dynamic range with its maximum

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at 1 mM. The PCNOID promoter was further evaluated regarding its induction dynamics.

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As shown in Figure 2C, it reached to its maximal induction after 6-8 hr, and

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maintained the expression level until the end of the experiments.

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Establishing an orthogonal gene expression system



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To further expand the genetic tools of C. testosteroni, we explored the possibility

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of creating a robust and specific gene expression system that is orthogonal to the host.

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To this end, we examined the T7 RNAP/PT7 system37 for potential applications in C.

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testosteroni. We first constructed the system by building two plasmids, pBRCM-

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T7RNAP and pEC01K-PT7. The former is based on the vector pCN00132 and carries

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T7 RNAP under the control of the PLAC promoter; the latter is based on the shuttle

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vehicle pEC01K and contains the GFP gene driven by the PT7 promoter (Figure 3A).

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Both the plasmids were then co-transformed into C. testosteroni PC, and the

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fluorescence production of the resulting strain was evaluated. As shown in Figure 3B,

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the cells containing the T7 RNAP/PT7 system indeed differentially produced

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fluorescence with the maximum occurring at 2 µM of IPTG induction, demonstrating

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that the orthogonal expression system indeed worked in C. testosteroni. Additionally,

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we noticed the leaky expression of the PT7 promoter in the absence of IPTG induction

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and reduced GFP expression when the IPTG level was 0.25 mM or higher, due to the

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toxicity of excessive T7 RNAP37.

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To facilitate the application of the T7 RNAP/PT7 system, we integrated the T7

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RNAP module into the C. testosteroni genome using the I-SceI based Calibrated

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Expression platform we recently developed. The specific procedure is following

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(Figure 3C): First, a landing pad, consisting of an intact kanamycin marker and an

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mKate2 reporter flanked by two I-SceI restriction sites, was integrated at a neutral

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locus between Ct_2988 and Ct_2989 to generate the receptor strain C. testosteroni R2.

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Second, the donor plasmid pICE102-T7RNAP, harboring the T7 RNAP gene under


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the control of PlacUV5 promoter, was constructed. Here, the T7 RNAP expression

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module was flanked by homogenous arms targeting the landing pad with I-SceI

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recognition sites on the both ends. Third, competent C. testosteroni R2/pICE102-

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T7RNAP cells were prepared. Fourth, the helper plasmid pICE101 expressing homing

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endonuclease I-SceI was transformed into the competent cells to introduce double

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stranded DNA breaks at both the landing pad in the chromosome and the donor

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plasmid, leading to the integration of the intact T7 RNAP cassette into the

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chromosome. Fifth, the helper plasmid pICE101 was eliminated through sucrose

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counter-selection.

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(Supplementary Figure S7), and named C. testosteroni T7R.

The

resultant

strain

was

confirmed

with

sequencing

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We evaluated the growth of C. testosteroni T7R/PT7 in response to different

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levels of IPTG induction (Supplementary Figure S8). We found that, at IPTG

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concentrations of 100 µM or less, no observable effects on cellular growth were

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observed. However, when the IPTG concentration is 250 µM or higher, cell growth

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was repressed. Figure 3D shows temporal GFP fluorescence dynamics. The system

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responded minimally upon 2.5 µM of IPTG induction, reached its maximum at 25 µM,

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and had the fastest response at 100 µM. An 18-fold inducible dynamic range was

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achieved. Notably, GFP expression was efficiently induced at the range of 10-100 µM

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IPTG.

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As previously reported, T7 RNAP/PT7 system had a leaky expression in the

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absence of IPTG, and caused an unnecessary burden to the host. To address this issue,

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we developed a different version of the T7 promoter by adding an additional Oid



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operator upstream of PT7 to enhance the LacI repression. Indeed, the resulting

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promoter, PT7DO (DO stands for Double Oid) (Figure 4A), was shown to reduce the

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leaky expression by up to 75% (Figure 4B). The corresponding temporal GFP

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fluorescence kinetics was shown in Figure 4C, where the dynamic response range was

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shifted towards higher IPTG levels compared to that of the original promoter (Figure

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3D). We also measured the growth of the cells harboring the new promoter, showing a

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similar pattern as those with the original promoters (Supplementary Figure S9).

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Constructing an orthogonal T7 promoter library

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Differential control of gene expression is critical for creating and fine-tuning

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gene networks such as biosynthetic pathways for desired functions. Thus, we were

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motivated to develop a library of inducible T7 promoters that span across a wide

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spectrum of response to IPTG induction. To create such a library, we utilized the

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engineered promoter PT7DO as the starting basis. The PT7DO promoter has an 18-bp T7

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core promoter region that can be divided into two domains, the T7 RNA polymerase

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binding domain and the transcription initiating domain38. Specifically, we introduced

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random mutations to the selected nucleotide sites of the promoter to alter its strength

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(Figure 5A). We then screened the library using fluorescence activate cell sorting

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(FACS) (Figure 5B), through which the top 0.15% of cells were collected and plated

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on solid agar. Subsequently, 200 colonies were picked to cultivate in culture with 100

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µM of IPTG induction, and the promoter strengths of the colonies were determined

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using a microplate reader. Finally, 22 variants whose promoter strengths span across


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the entire spectrum were selected to form the promoter library PT7DOM (PT7DO mutants).

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The specific promoter sequences of the library is shown in Supplementary Table S1.

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We evaluated the activity of the PT7DOM library in response to different levels

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of IPTG induction (Figure 5C). Of note, at the level of 100 µM IPTG induction, 239-

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fold variations were achieved, with the highest by the promoter PT7DO and the lowest

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by PT7DOM1. Associated with the library, Figure 5D illustrates the relative nucleotide

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abundance at individual nucleotide sites using the sequence logo39-40. In the library,

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the promoter PT7DOM22 is the strongest promoter that has no detectable leakage. We

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thus characterized its response characteristics (Figure 5E), showing an efficient

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expression upon 50-250 µM of IPTG induction. Together, we successfully established

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a T7 promoter library with differential gene expression kinetics for C. testosteroni for

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fine tuning of engineered gene networks.

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Conclusion

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The development of next-generation cellular chasses is the key to move synthetic

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biology from laboratory demonstrations to field applications. C. testosteroni is a

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promising chassis which allows for robust performance in complex field

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environments. To facilitate the realization of the full potential of C. testosteroni

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engineering, in this work, we developed a synthetic biology toolbox that is composed

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of E. coli-C. testosteroni shuttle vehicles and a set of well-refined promoters.

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Our newly built shuttle vehicle allows rapid genetic circuit building in E. coli, by

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coupling traditional recombinant DNA technique with recent cloning methods such as



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Gibson assembly, and quick phenotypic validation by directly transforming resulting

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circuit-carrying plasmids into C. testosteroni, thereby enriching the vector repertoires

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for C. testosteroni, similar to recent efforts in pathway engineering in lactic acid

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bacteria46. Besides the vectors, promoters are another class of fundamental DNA parts

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for gene circuit development. We thus tested four inducible promoters adopted from E.

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coli, designed two inducible promoters with hybrid structures, constructed an

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orthogonal T7 RNAP/PT7 based expression system, and further established a library of

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T7 promoters with differential induction characteristics. Together, these promoters

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and expression systems significantly expand our ability to control gene expression in

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C. testosteroni with desired induction spectrum and dynamics. Notably, our synthetic

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T7 promoters are strong and sensitive to IPTG induction. For instance, we observed

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the promoter in Fig. 3D has noticeable GFP expression with the IPTG concentration

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as low as 0.0025 mM and has the strongest expression at 0.025 mM. However, the

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GFP expression decreases with further increase of IPTG and stopped upon the

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induction of 0.25 mM or higher IPTG. We speculate that, the loss of GFP expression

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at high IPTG levels are due to excessive GFP production which may cause the

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formation of inclusion bodies47.

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Creating tools for non-model cellular chasses are the fundamental prerequisite

300

for synthetic biology to address real-world challenges. The toolkit developed in this

301

study provides a valuable platform for rapid and effective engineering of gene circuits

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in C. testosteroni, facilitating the application of the organism for bioremediation

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purposes. For example, the toolkit will enable us to construct and systematically


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optimize de novo pathways for mediating refractory aromatic pollutants including

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industrial chemicals, pesticides and pharmaceuticals.

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As our primary goal of the study to develop tools for engineering C. testosteroni,

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we cured the plasmid pCNB1 to increase the flexibility of plasmid transformation and

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reduce the metabolic cost associated with the mega-size plasmid. Meanwhile, its

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elimination is accompanied with the removal of the 4-chloronitrobenzene pathway. In

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the future, if needed, 4-chloronitrobenzene remediation can be restored by introducing

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the pathway into our shuttle system or integrating into the chromosome.

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As a first step to establish C. testosteroni as a useful microbial chassis, this study

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also sheds light on future developments of relevant genetic tools for the organism. We

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speculate that broad-host-range vectors and promoters in other gram-negative bacteria

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can be potentially adapted for C. testosteroni with proper modifications. For instance,

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IncP-1β type plasmids, previously explored as shuttle plasmids in other organisms,

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such as in Pseudomonas putida41, are worthy of exploration for propagation in C.

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testosteroni. In addition, as the IncP-1β based shuttle systems is compatible with the

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broad host vector pBBR142, it will be feasible to enable stable propagations of two

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plasmids. Besides the gene expression tools developed in the study, latest genome

321

editing tools such as CRISPR-Cas9/Cpf1 system43-45 shall be developed for C.

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testosteroni in the future to further facilitate it as synthetic biology chassis in

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environmental application.

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Materials and Methods

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Strains, plasmids, and cultivation conditions

327

The bacterial strains and plasmids used in this study are listed in Table 1. E. coli

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NEB10β was used for general plasmid construction, and C. testosteroni PC was used

329

for phenotypic validation. All strains were cultivated aerobically at 37 °C (E. coli) or at

330

30 °C (C. testosteroni) in Luria-Bertani (LB) medium with 200 rpm shaking or LB

331

plates with 1.5% (w/v) agar. L-arabinose metabolism experiment was performed using

332

L-arabinose as the sole carbon source in the mineral salt basic (MSB) medium25 and

333

using MSB medium supplemented with 20 mM of sodium succinate as the positive

334

control. When necessary, appropriate antibiotics were added at the following

335

concentrations (µg/mL): for E. coli: 100 of Ampicillin; 50 of kanamycin, 20 of

336

chloramphenicol, 10 of tetracycline; for C. testosteroni, 200 of kanamycin, 25 of

337

chloramphenicol, 20 of tetracycline. Inducers of L-arabinose and IPTG were added to

338

the medium at the concentrations indicated. Sucrose was added at 20% (w/v) for

339

counter-selection.

340

Genetic manipulation and transformation

341

Plasmid and chromosomal DNAs were isolated using the E.Z.N.A Plasmid Mini

342

Kit and the E.Z.N.A Bacterial DNA kit (OMEGA, Beijing, China). DNA fragments for

343

plasmid assembly were purified using the E.Z.N.A Gel Extraction Kit (OMEGA,

344

Beijing, China). PCR enzymes, Gibson mix enzymes and restriction enzymes were

345

purchased from New England BioLabs (U.K.). Q5 High-Fidelity DNA Polymerase was

346

used for amplifying fragments for DNA assembly. PCR verification was performed


Page 16 of 41

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347

with OneTaq 2X Master Mix. DNA sequencing was performed by Genewiz (Beijing,

348

China).

349

E. coli electroporation was performed as previously described46; electroporation

350

for C. testosteroni was adapted from protocols for a wide range of gram-negative

351

bacteria48-51. Both of E. coli and C. testosteroni were prepared for electro-competent

352

cells by washing twice with ice-cold 10% glycerol, and concentrating 100-fold.

353

Electroporation was performed with pre-chilled 2 mm gap electroporation cuvettes

354

(Bio-Rad) and electroporated at 2.5 kV with a Bio-Rad MicroPulser. One mL of LB

355

was added to shocked cells and recovered for 1 (E. coli) and 2 hrs (C. testosteroni),

356

before plating on LB agar with appropriate antibiotics.

357

Plasmid construction

358

The Gibson assembly method52 was used for construction of plasmids. Primers

359

used in this study are listed in Table S2. The p15A origin was amplified from plasmid

360

pNZ5319, and assembled with the kanamycin cassette amplified from plasmid

361

pBBR1-MCS2 to generate plasmid p15AKan. The trfA1 and oriV were amplified

362

from C. testosteroni CNB-1 total DNA and cloned into plasmid p15AKan,

363

respectively, to generate plasmid p15AKan-trfA and p15AKan-oriV. The trfA1 and

364

oriV were then amplified and cloned into plasmid p15AKan to construct plasmid

365

pEC01K. Multiple cloning sites was introduced on primers MCS-P15KAN F and

366

MCS-ORIV R. The plasmid pEC01K kanamycin marker was replaced with a

367

tetracycline resistance gene amplified from plasmid pITK to generate plasmid

368

pEC01T.



369

In order to assemble inducible promoters from E. coli to utilize in C. testosteroni

370

PC, they were first assembled to the plasmid pEC01K. Specifically, the plasmid

371

backbone was digested for linearization, the consensus parts were amplified with

372

blunt primers resulting in a blunt end, and the different inducible promoter blocks

373

were amplified with overlap ends with adjacent parts. Finally, the prepared genetic

374

elements were assembled in one-step with the Gibson method. Plasmid pEC01K was

375

linearized to prepare the plasmid backbone. The GFPmut3b gene was amplified with

376

pCP202; double terminator T1T2 was amplified from plasmid pITK. Then, the araC-

377

PBAD cassette was amplified from plasmid pKD4653 and assembled with linearized

378

pEC01K, amplified GFPmut3b and T1T2 to generate plasmid pEC01K-PBAD; the lacI-

379

PLAC was amplified from plasmid pITK and assembled with the other three elements

380

to generate plasmid pEC01K-PLAC; the lacIQ-PTAC cassette was amplified from

381

plasmid pXMJ19 and assembled with the other elements to generate plasmid

382

pEC01K-PTAC; the lacIQ-PTRC was amplified from plasmid pTrc99a and assembled

383

with the other elements to generate plasmid pEC01K-PTRC.

384

To construct the hybrid promoter pEC01K-PCNOID, the promoter PCN was

385

amplified from plasmid pCP202. An Oid operator was introduced on the reverse

386

primer; the lacI cassette was amplified from plasmid pITK; they were then assembled

387

with GFP, T1T2 and the linearized pEC01K to generate plasmid pEC01K-PCNOID. A

388

second Oid operator was then introduced through inverse PCR amplification of

389

pEC01K-PCNOID to generate plasmid pEC01K-PCN2OID.


Page 18 of 41

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390

To validate the T7RNAP/PT7 system, the T7 RNAP cassette was amplified from

391

the E. coli BL21(de3) genome, and cloned into plasmid pCN001 to generate plasmid

392

pCN001-T7RNAP. The landing pad integration plasmid, pNZ-I-SceI-PAD, was

393

constructed as follows: The primer pair PNZ-UP F/KAN-UP R and KATE-DOWN

394

F/PNZ-DOWN R was used to amplify the two homologous arms respectively; the

395

primer pair UP-KAN F/KATE-KAN R was used to amplify the kanamycin cassette

396

from pBBR1-MCS2; the primer pair KAN-MKATE F/DOWN-MKATE R was used

397

to amplify mKate gene from the plasmid pTX-MKATE; and the primer pair BLT-

398

PNZ F/R was used to amplify the PNZ5319 backbone. The five fragments were then

399

assembled together to generate the plasmid pNZ-I-SCEI-PAD. The donor plasmid for

400

T7 RNAP integration was built by amplifying 1-kb upper and lower arms of the

401

integration site with C. testosteroni CNB-1 DNA as the template. They were

402

assembled with the T7 RNAP cassette into plasmid pICE102 to generate donor

403

plasmid pICE102-T7RNAP. The homing endonuclease I-SceI restriction sites were

404

introduced to flank the whole structure. To construct plasmid pEC01K-PT7, the

405

promoter PT7 and the T7 terminator were both amplified from plasmid pET-19b; the

406

reporter gene GFPmut3b was amplified from plasmid pCP202; terminator TET was

407

amplified from pCP202; they were cloned into linearized pEC01K to generate

408

plasmid pEC01K-PT7. The mutant PT7DO encoding plasmid pEC01K-PT7OID was

409

constructed by inverse amplifying pEC01K-PT7 and introducing an Oid operator

410

upstream of the T7 promoter sequence. The PT7DOM promoter library was constructed



411

by inverse PCR amplification of pEC01K-PT7DO to use random sequence

412

“NNNNNCGACTCNNTATAGG” to replace the native sequence.

413

Plasmid transformation efficiency and stability assessment

414

The plasmid transformation efficiency was calculated by transforming 1 µg

415

plasmid DNA into 50 µl of cells. After recovery, the culture was diluted and plated on

416

LB plates with corresponding antibiotics. After 24–36 hr of incubation at 30 °C,

417

formed colonies were counted.

418

C. testosteroni PC/pEC01K was grown for 100 generations in LB medium with or

419

without the antibiotics. It was then diluted and plated on LB agar plates without

420

antibiotics. Colonies were subsequently picked for antibiotic-resistant tests. A total of

421

one hundred colonies were tested (three replicates).

422

T7 RNAP genome integration

423

To integrate T7 RNAP into the genome, the receptor strain C. testosteroni R2 was

424

first constructed. The plasmid pNZ-I-SceI-Pad was transformed into C. testosteroni

425

PC. The primer pair, Con.LA_UP F/ SEQ.KAN R and SEQ.MKATE F/Con.RA_DW

426

R, was used to screen for the double crossover events. Receptor strain C. testosteroni

427

R2 was transformed with donor plasmid pICE102-T7RNAP and prepared for

428

competent cells. It was then transformed with I-SceI expression helper plasmid

429

pICE101 and induced for its expression. The homing endonuclease I-SceI cleaved the

430

donor plasmid and the landing pad, stimulating homologous recombination, resulting

431

in the integration of T7 RNAP cassette. The primer pair Con.LA_UP F/ Con.T7R R

432

and Con.T7R F/ Con.RA_DW R was used to confirm the integration (Supplementary


Page 20 of 41

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433

Figure S7A). The corresponding PCR products were sequenced for further

434

confirmation (Supplementary Figure S7B). The donor plasmid pICE102-T7RNAP

435

was cured by I-SceI cleavage and the helper plasmid pICE101 was finally cured

436

through counter-selection on plate supplement with 20% sucrose.

437

Growth and promoter strength measurements

438

Cell growth and the strengths of the promoter driving GFPmut3b were measured

439

using a microplate reader. Overnight bacterial cultures harboring corresponding

440

plasmids were sub-cultured 1% (v/v) into fresh LB medium. When OD600 was

441

reached ~0.2, 200 µL of them were added into wells of a 96-well flat clear bottom

442

plate (Corning Costar, cat. # 3603). It was then put on the microplate reader (BioTek,

443

SynergyH4) for growth (detected at 600 nm (OD600)) and GFP fluorescence

444

(excitation wavelength of 488 nm; emission wavelength of 520 nm) measurement.

445

Notably, due to the strength difference of different promoters, different gains were

446

used across figures. The plate was covered with lid and sealed with parafilm to

447

minimize evaporation. The promoter using LacZ as the reporter was evaluated by

448

mixing 50 µL cell culture with 5 µL X-gal solution (20 mg/mL).

449

To evaluate the decrease of leaky expression of promoter PT7DO compared to the

450

wild-type PT7 promoter, both the PT7DO and PT7 promoters were measured at the

451

absence of IPTG induction. The repression range was calculated as (PT7-PT7DO)/PT7.

452

FACS screening

453 454

The PT7DOM promoter library of varying strength was generated from a random library.

After

inverse

PCR

amplification

to

introduce


random

sequence


455

“NNNNNCGACTCNNTATAGG”, the PCR product was cyclized through Gibson

456

and transformed into E. coli NEB10β. When grown up, the colonies formed were

457

scraped from the plate and extracted for the plasmids. The mixed plasmids were used

458

to transform strain C. testosteroni T7R, and induced with 25 µM IPTG. FACS

459

screening was performed to select the functional promoters with the BD FACSCalibur

460

Flow Cytometer with a 488-nm excitation laser and the FL1 (530/30 nm band-pass

461

filter) detector. The top 0.15% strength was screened out. The recovered cells were

462

plated on agar plate for growth. Two hundred colonies were chosen for microplate

463

reader analysis. Promoters of varying strengths were selected to make up of the

464

PT7DOM library.

465


Page 22 of 41

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466

ASSOCIATED CONTENT

467

Supplementary Materials

468

Figure S1. Schematic for the construction of the E. coli-C. testosteroni shuttle

469

vehicles.

470

Figure S2. Growth curves for the strain C. testosteroni PC, C. testosteroni PC with

471

the plasmid pEC01K and C. testosteroni PC with the plasmid pEC01T.

472

Figure S3. Growth patterns of the strain C. testosteroni PC with different promoters

473

and growth media.

474

Figure S4. Evaluation of the promoter strength through X-gal staining.

475

Figure S5. Promoter sequences of the two hybrid promoters.

476

Figure S6. Growth curve of C. testosteroni PC/pEC01K-PCNOID in response to

477

different concentrations of IPTG

478

Figure S7. Validation of the genomic integration of T7 RNA polymerase in C.

479

testosteroni PC.

480

Figure S8. Growth patterns of C. testosteroni T7R/pEC01K-PT7 in response to

481


482

Figure S9. Growth patterns of C. testosteroni T7R/pEC01K-PT7OID in response to

483


484 485

Table S1: PT7DOM promoter library variants

486

Table S2: Primers used in this study

487



488

AUTHOR INFORMATION

489

Corresponding Author

490

Shuang-Jiang Liu (Tel: +86-10-64807423. Fax: +86-10-64807421, Email:

491

[email protected]), and Ting Lu (Tel: +1-217-333-4627. Fax: +1-217-265-0246. Email:

492

[email protected])

493

Notes

494

The authors declare no competing financial interest.

495

Acknowledgements

496

We thank Dr. Zhao Tong for aid of performing FACS screening, and Dr. Howard

497

Gelberg for editing the manuscript. This work was supported by National Natural

498

Science Foundation of China (NSFC grant No. 31230003) to S-J Liu and

499

CAS/SAFEA international partnership program to S-J Liu and T Lu. This work was

500

also supported in part by the National Science Foundation (No. 1553649 and 1227034)

501

to T. Lu.

502

Author contributions

503

Shuang-Jiang Liu and Ting Lu conceived this project and supervised the study. Qiang

504

Tang designed and conducted the experiments. Qiang Tang, Ting Lu and Shuang-

505

Jiang Liu analyzed the data. Qiang Tang, Ting Lu and Shuang-Jiang Liu wrote the

506

manuscript.

507


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49. Huang, W., and Wilks, A. (2017) A rapid seamless method for gene knockout in Pseudomonas aeruginosa. BMC Microbiol, 17, 199.

653

50. Martinez-Garcia, E., and de Lorenzo, V. (2012) Transposon-based and plasmid-

654

based genetic tools for editing genomes of gram-negative bacteria. Methods Mol

655

Biol, 813, 267-283.



656

51. Hülter, N., Sørum, V., Borch-Pedersen, K., Liljegren, M. M., Utnes, A. L. G.,

657

Primicerio, R., Harms, K., and Johnsen, P. J. (2017) Costs and benefits of natural

658

transformation in Acinetobacter baylyi. BMC Microbiol, 17, 34.

659

52. Gibson, D. G., Young, L., Chuang, R. Y., Venter, J. C., Hutchison, C. A., Ⅲ,

660

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

661

hundred kilobases. Nat Methods 6, 343-345.

662

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

663

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

664

97, 6640-6645.

665

54. Lambert, J. M., Bongers, R. S., and Kleerebezem, M. (2000) Cre-lox-based

666

system for multiple gene deletions and selectable-marker removal in

667

Lactobacillus plantarum. Appl Environ Microbiol 73, 1126-1135.

668


Page 32 of 41

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669


Table 1 strains and plasmids used in this article Strains or plasmids

Description

References

Strains E. coli NEB10β

∆(ara-leu) 7697 araD139 fhuA ∆lacX74 galK16 galE15 e14- ϕ80dlacZ∆M15 recA1 relA1 NEB

E. coli BL21(DE3)

E. coli str. B F– ompT gal dcm lon hsdSB(rB–mB–) λ(DE3 [lacI lacUV5-T7p07 ind1 sam7 Lab stock nin5]) [malB+]K-12(λS)

C. testosteroni CNB-1

wild-type

23

C. testosteroni PC

a plasmid-cured derivative of C. testosteroni CNB-1

This study

C. testosteroni R2

KanR-mKate2 landing pad integrated at locus between Ct_2988 and Ct_2989

This study

C. testosteroni T7R

lacI-PlacUV5-T7 RNAP integrated at locus between Ct_2988 and Ct_2989

This study

Broad host range plasmid compatible with IncQ, IncP, IncW, and colE1; KanR

42

Plasmids pBBR1-MCS2 pCN001

pBBR1 origin, Cm

32

R

pNZ5319

p15A, Cm , Em , lox66, lox71 recombination sites

54

pEC01K

p15A, KanR, trfA, oriV, multi genetic circuit insertion sites

This study

pNZ-I-SceI-PAD

pNZ5319 derivative, containing I-SceI-Kan-mkate-I-SceI landing pad

This study

pITK

TetR, lacIQ-PLAC

Lab stock

pTX-MKATE

RepA, P3A-MKATE

Lab stock

pCP202

pBBR1, PCN-GFP

This study

pEC01T

p15A, TetR, trfA, oriV, multi genetic circuit insertion sites

This study

p15AKan

p15A, KanR

This study

R

This study

R

R

p15AKan-trfA

p15A, Kan , trfA1

p15AKan-oriV

p15A, KanR, oriV

This study

pKD46

pSC101, AmpR, araC-PBAD-lambda red recombinase

52

pEC01K-PBAD

p15A, KanR, oriV, trfA, araC-PBAD-gfpmut3b-T1T2

This study

pEC01K-PLAC

p15A, KanR, oriV, trfA, lacI-PLAC-gfpmut3b- T1T2

This study

pXMJ19

pBL1 oriV, pUC origin, CmR, lacIq, PTAC,

University of Bielefeld

pEC01K-PTAC

p15A, KanR, oriV, trfA, lacIQ-PTAC-gfpmut3b- T1T2

This study

Q

R

pTrc99a

lacI -PTRC, Amp

Lab stock

pEC01K-PTRC

p15A, KanR, oriV, trfA, lacIQ-PTRC-gfpmut3b-T1T2

This study

pEC01PCNOID

p15A, KanR, oriV, trfA, lacI-PCN-Oid-gfpmut3b-T1T2

This study

R

pEC01PCN2OID

p15A, Kan , oriV, trfA, lacI-PCN-2Oid-gfpmut3b-T1T2

This study

pICE102

p15A, TetR, trfA, oriV, donor plasmid

This study

pICE102-T7RNAP

pICE102 derivative plasmid, carrying IPTG inducible T7 RNA polymerase, used for T7 R

Q

This study

pICE101

pBBR1-MCS2 derivative plasmid, Cm , lacI -PCNOID-I-SceI, sacB

pCN001-T7RNAP

pBBR1 origin, CmR, carrying IPTG inducible T7 RNA polymerase

This study

pEC01K-PT7

p15A, KanR, oriV, trfA, PT7-Oid-GFPmut3b-T7 terminator

This study

pEC01K-PT7OD

p15A, KanR, oriV, trfA, Oid-PT7-Oid-GFPmut3b-T7 terminator

This study

pEC01K-PT7DOM1-22

Member of the PT7DOM promoter library

This study

670 671


This study


672

Figure Legends

673 674

Figure 1. A shuttle system of E. coli-C. testosteroni for genetic cloning and gene expression. (A)

675

Schematic of the E. coli-C. testosteroni shuttle system. (B) The plasmid map of pEC01K and pEC01T.

676

(C) Scheme of multiple genetic element assembly. The plasmid backbone was linearized through

677

digestion. Different inducible promoters and the consensus parts were amplified through PCR. All of

678

the genetic elements were later assembled using Gibson assembly. (D) Characterization of the

679

inducible promoter systems. Left panel: lacIQ-PTAC; Middle panel: lacIQ-PTRC; Right panel: araC-PBAD.

680

(RFU: relative fluorescence unit). Three replicates were used to determine the means (bars) and

681

standard deviations (error bars).

682 683

Figure 2. Hybrid IPTG-inducible promoters. (A) Schematic of two hybrid IPTG-inducible

684

promoters.

685

Upper: the PCNOID promoter with one Oid operator site; Lower: the PCN2OID promoter involving two

686

Oid operator sites. (B) Evaluation of the hybrid promoters PCNOID (squares) and PCNOID (triangles)

687

under IPTG inductions. Green fluorescence was detected at 18 hr. Three replicates were used to

688

determine the means (symbols) and standard deviations (error bars). (C) Heat map of green

689

fluorescence protein production induced by the promoter PCNOID at various IPTG concentrations.

690 691

Figure 3. An orthogonal expression system based on T7 RNAP/PT7. (A) The plasmid map of pEC01K-

692

PT7 and the PT7 promoter structure. (B) Evaluation of the T7 RNAP/PT7 expression system. Three

693

replicates were used to determine the means (bars) and standard deviations (error bars). (C) Schematic


Page 34 of 41

Page 35 of 41 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


694

for the integration of T7 RNAP into the chromosome. (D) Evaluation of C. testosteroni T7R/pEC01K-

695

PT7 at various IPTG concentrations. Three replicates were used for each experiment.

696 697

Figure 4. The PT7DO mutant promoter with reduced leaky expression. (A) The design of the

698

PT7DO promoter. (B) Comparison of the leaky expression by the engineered PT7DO promoter (circles)

699

and PT7 (squares) promoters. Here, the expression was normalized by the leakage of PT7. (C) Heat

700

map of green fluorescence protein production by the PT7DO promoter under varied IPTG

701

concentrations.

702 703

Figure 5. Establishing the PT7DOM promoter library for C. testosteroni. (A) Random nucleotide

704

sequences used to construct the library. (B) The procedure to construct and select the PT7DOM promoter

705

library. (C) Green fluorescence protein production by the variants of the PT7DOM library. Three

706

replicates were used to determine the means (symbols) and standard deviations (error bars). (D)

707

Sequence logo of the orthogonal PT7DOM promoter library. The sequence logo was constructed using

708

WebLogo44-45. (E) Heat map of green fluorescence protein production by the promoter PT7DOM22 under

709

varied IPTG concentrations over time. Three replicates were used for each experiment.

710


Genetic Toolkit ACS Synthetic Biology

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

Page 36 of 41

C. testosteroni

Shuttle vehicle

Multi-part assembly ACS Paragon Plus Environment

Promoter library

A.

C. testosteroni Genetic Circuit

Shuttle Vehicle

p15A O.R.pCNB1

Vector preparation

pEC01K/T MCS

araC lacI

Digestion

lacIQ

o ri V

Element amplification

PBAD

Over-lap ends

MCS site

PLAC Blunt-ends

PTAC

gfpmut3b

T1T2

PTRC

lacIQ

gfpmut3b

D.

T1T2

RFU

RFU

IPTG (mM) 0 0.25 0.5 1 2.5 5 10

IPTG (mM) 3500 0 1 2.5 5 2500 10

300 200 100 0

e.g. Phenotypic validation, pollutant degradation, etc.

E. coli

C.

p15A

trfA1

e.g. Genetic circuit construction, Promoter tuning, etc.

Marker

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 55 500 56 57 58 400 59 60

B.


Tet R Ka nR

Page 37 of 41

6 hr

12 hr

lacI -PTAC Q

18 hr

RFU

2500 1500

500

500

6 hr

12 hr

18 hr

ACS Paragon Plus Environment

lacI -PTRC Q

0 5 10 50 100

3500

1500

0

L-arabinose (mM)

0

6 hr

12 hr

18 hr

araC-PBAD

A.

LacI

GFPmut3b

Oid

PCNOID LacI

T1T2

RFU

10

PCN2OID

2

3

0.5

1.0x10

0.1

0.025

2.0x102 0.0

2

3.0x10


Time (hr)

24

0 0.001 0.005 0.010 0.025 0.050 0.10 0.25 0.5 1 2.5 5 10

0.005

IPTG (mM)

3

1.8x10

2.5

18

1.0x103

PCNOID

12

RFU

1.4x103

6.0x10

T1T2

C.

3

6

1.8x10

Page 38 of 41

PCN2OID

0

B.

GFPmut3b

IPTG (mM)

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


6.0x10 3

Oid

T7 RNAP

I-

Ct_2989

Strain T7R


Curing

Time (hr)

2

1

0.5

0.1

0.25

18

lacI

PlacUV5

pICE101

3.1x103

15

Plac

Transform of helper plasmid

Receptor strain R2

1.6x104

9.6x103

12

KanR mKate2

pICE102-T7RNAP

1 0.5 0.25 0.1 0.05 0.025 0.01 0.005 0.0025 0.001 0

9

I-SceI

T7 RNAP

RFU

0

Ct_2988 Chr

lacI

IPTG (mM)

IPTG (mM)

Chr

PlacUV5

eI

I-SceI

Plac

D.

Sc

C.

0.05

T7 terminator AGTAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAA

0.025

0.0

0.01

TCGATCCCGCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTC RBS CCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACC GFP TACTAG

0.005

2.0x10 3 0.002

T7 promoter

6

Kan

4.0x10 3

0.001

5

T7-ter

R

3

p1 A

8.0x10 3

RFU

pEC01K-PT7

1.0x10 4

PT7

GFP

trf A 1

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

B.


0

A.

oriV

Page 39 of 41

A.

Page 40 of 41

GGAATTGTGAGCGGATAACAATTCCTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCC

100

0

3

Oid

Time (hr) 6

9

12

15

PT7 50

18

Leakage reduction

PT7DO

Ti

0.0 RFU

1 0.5 0.25 0.1 0.05 0.025 0.010 0.005 0.0025 0.001 0

1.6x104

9.6x103


Time (hr)

18

15

12

9

6

3.2x103

3

0

C.

Relative Leaky expession (%)

B.

PT7

Oid

IPTG （mM）

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


Page 41 of 41 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



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