Rapid Diversification of BetI-Based Transcriptional Switches for the

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The rapid diversification of BetI-based transcriptional switches for the control of biosynthetic pathways and genetic circuits. Kazuya Saeki, Masahiro Tominaga, Shigeko Kawai-Noma, Kyoichi Saito, and Daisuke Umeno ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.5b00230 • Publication Date (Web): 18 Mar 2016 Downloaded from http://pubs.acs.org on March 27, 2016

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ACS Synthetic Biology

The rapid diversification of BetI-based transcriptional switches for the control of biosynthetic pathways and genetic circuits. Kazuya Saeki1, Masahiro Tominaga1, Shigeko Kawai-Noma1, Kyoichi Saito1, and Daisuke Umeno1, 2, * 1

Department of Applied Chemistry and Biotechnology, Faculty of Engineering, Chiba University, 1-33 Yayoi-Cyo, Inageku, Chiba 263-8522, Japan 2 Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency (JST), 41-8 Honcho, Kawaguchi, Saitama 332-0012, Japan

KEYWORDS: induction system, genetic switch, directed evolution, selection, liquid-handling, carotenoid, lycopene, operon, Boolean logic gates ABSTRACT: Synthetic biologists are in need of genetic switches, or inducible sensor/promoter systems, that can be reliably integrated in multiple contexts. Using a liquid-based selection method, we systematically engineered the choline-inducible transcription factor BetI, yielding various choline-inducible and choline-repressive promoter systems with various input-output characteristics. In addition to having high stringency and a high maximum induction level, they underwent a graded and single-peaked response to choline. Taking advantage of these features, we demonstrated the utility of these systems for controlling carotenoid biosynthetic pathway and for constructing two-input logic gates. Additionally, we demonstrated the rapidity, throughput, robustness, and costeffectiveness of our selection method, which facilitates the conversion of natural genetic controlling systems into systems that are designed for various synthetic biology applications.

INTRODUCTION: Transcriptional induction systems, consisting of promoters, operators, regulators and inducers, have been widely used as indispensable tools in the field of metabolic engineering and synthetic biology.1-4 Despite their great success in creating diverse types of synthetic networks,5 they are constructed of surprisingly few genetic components. Efforts to increase the number of available genetic parts,6-11 as well as to improve the predictability of the behavior of these parts,12 are in progress.13 In contrast, systematic efforts to rebuild existing switches/devices are few. Even the simplest organism (prokaryote) has hundreds of transcription factors; there are 199 entries for Escherichia coli transcriptional controlling proteins in EcoCyc.14-17 This number is already sufficiently large for any conceivable synthetic biology project. Unfortunately, however, most of these sensor/promoter systems cannot be directly used for synthetic biology applications because of their inappropriate switching properties. For instance, the switching thresholds of genetic switches are programmed by evolution to have physiological relevance, and the induction of such systems almost always elicits physiological responses. Thus, the sensitivity of the sensory proteins must be modified so that they undergo ON/OFF transition at a lower inducer concentration than would ordinarily exert a physiological influence. Genetic switches, regardless of their physical implementations or their mechanisms of action, ultimately turn gene expression ON or OFF under well-defined conditions. Thus, directed evolution can be applicable to any genetic switch simply by harnessing the positive/negative selectors under

their control. A variety of unique selection systems18-27 have been developed. For the development of our selection systems, we emphasized the following two features. First, the entire selection should be completed solely by liquid handling. This is particularly important for realizing the automated selection processes and to ensure throughput. Second, the selection process should be completed rapidly. This is especially important for multi-state genetic circuits, which require many ON/OFF selections. To this end, we chose and optimized both ONselection and OFF-selection, which were decoupled from cell growth.28 In this work, we applied our latest selection platform28 to conduct directed evolution of BetI-based switches for the robust and precise control of metabolic pathways and genetic circuits. Recently, we have engineered choline-inducible T7 promoter by modifying T7 promoter and Escherichia coli protein BetI29. By down-tuning the BetI-operator affinity, modifying the sequence to reduce the putative secondary structure of the transcript, we could achieve unprecedentedly high expression level of gene under its control. This system was designed sorely for protein over-expression, and activation of the operon was lethal to the host cell. Therefore, we decided to freshly conduct directed evolution of BetI-based switches to well adapt for the robust and precise control of metabolic pathways and genetic circuits. To develop a set of choline-inducible/repressible promoter systems, a single round of rapid, seamless, and parallel operation of ON/OFF selection was conducted with a BetI library generated by error-prone PCR. Diversity in isolated switching properties, as well as the

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negligible appearance of false positives, underscored the power of our selection platform. Additionally, high stringency, high maximum induction level, portability, the graded/monodispersed response to cheap and safe inducer choline, and plug-and-playability in various contexts were demonstrated for the resulting switches. Together with the recently developed choline-inducible T7 promoter systems tailored for protein overexpression,29 the BetI-based switches presented in this work will be a promising tool for metabolic engineering and synthetic biology. RESULTS AND DISCUSSION: BetI-regulated T5 promoter system. A variety of bacteria possess choline metabolizing Bet operons. In E. coli, the expression of bet genes is negatively regulated by BetI, a TetR family repressor, via specific binding to the bet operator (betO).30,31 Upon binding with choline, BetI falls off of betO, thereby allowing the transcription of the genes downstream from the operator.31 We synthesized the 20-nucleotide sequence (5’-TTAATTGAACGTTCAATTAA-3’), annotated as betO in EcoCyc,14-16 directly next to the -10 sequence of the T5 promoter (Figure 1). To control this T5P/betO maintained on the pBR322-based (pMB1-based) plasmid, we created a pACYC-based (p15A-based) plasmid carrying the gene encoding E. coli BetI. It should be noted that in addition to this plasmid-borne betI, there is another copy of betI on the chromosome. To ensure the utility in various E. coli strains, we decided to evolve Bet switches without removing the chromosomal betI. To ensure the stoichiometric dominance of the plasmid-encoded BetI mutants over the chromosomal counterpart, we assigned a highly active promoter (PC132) and efficient ribosome binding site (72,659 in rbs score33) to betI (and its mutant library). To measure the expression level of the resulting T5P/betO, the reporter gene sfgfp34 was placed under this promoter. Further downstream, we placed hsvTK-aph28, which was recently developed for the rapid, liquid-based selection of the ON/OFF states of the genetic switches.

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cent reporter for the state of Bet-switches, while hsvtk::aph is used for functional selection of the Bet-switches 28. hsvTK acts as the negative selector (OFF-selector) for removing the ON-state switch variants upon adding artificial nucleoside dP, 26 and aph (kanamycin resistance gene) acts as the positive selector (ON-selector) for removing OFF-state variants.

When the plasmid pT5/betO-sfgfp-hsvtk::aph was introduced, E. coli strains exhibited bright fluorescence both in the presence and absence of choline in the medium (not shown). This indicated that the chromosomal expression of BetI was insufficient to cover the plasmid-coded betO loci. By additionally transforming E. coli with plasmid-coding wild-type BetI (BetIWT), we found that the transformant cells exhibited virtually no fluorescence in all tested choline concentrations. Starting from the behavior of the prototype system, an “always-OFF” switch, we sought to evolve it into functional choline-inducible or choline-repressive switches without loss of stringency. Parallel selection for functional Bet-ON/Bet-OFF mutants. Recently, we have isolated BetI variants that control pT7-based protein overexpression systems29 using GFP-based screening. Because the functions of genetic switches are highly context dependent and because a recently developed selection platform28 enables the rapid and robust operation of multiple directed evolution experiments in parallel, we decided to freshly select for functional Bet-ON/Bet-OFF transcriptional switches, this time for the promoter fueled by E. coli RNA polymerases (Figure 2A). Thus, we could eliminate the dependence on the foreign (T7) polymerase, improving the portability of the system. The first step of the directed evolution experiment was to introduce random mutations into betI. To this end, we amplified the entire reading frame of betI in a mutagenic condition (error-prone PCR). The resulting PCR product was subcloned back into the expression vector, resulting in pC1-[betI]mut (square brackets indicate it is PCR-randomized, library size ~ ca. 2 × 105). The pC1-[betI]mut was electroporated into the cell harboring pT5/betO-gfp-hsvtk::aph, and the resultant cells were subjected to flow cytometric analysis. Approximately 60% of the cells were non-fluorescent (phenotypically wild type-like) (Figure 2B (b)). Approximately 40% of the transformant cells fluoresced in the absence of choline. It was assumed that most of them were those harboring ‘dead’ mutants that were impaired either in operator binding or in folding. Based on our previous work,29 we knew that a minor but certain fraction of the fluorescent cells would harbor BetI mutants with reversed phenotype. This distribution was almost unchanged even in the presence of 100 mM choline, indicating that the majority of the mutant BetI in this initial library pool were either wildtype-like (always-OFF) or broken (alwaysON), and the fraction of the reversed mutants was much smaller.

Figure 1. The T5P/betO-based transcriptional controlling system used in this work. Under the control of BetI-regulated promoter, sfGFP and hsvtk::aph are placed. sfGFP is fluores-


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Figure 2. Parallel operation of OFF-/ON-selections to isolate choline-inducible (Run-1) and choline-repressive (Run-2) transcriptional switches. (A) The experimental workflow for selecting for choline-inducible/choline-repressive switches. (B) Flow cytometric analysis of cell population harboring BetI mutant plasmids and probe/selector-plasmid in each round of section processes. Distribution in fluorescence signal is given in the presence (shown in red) and in the absence (shown in gray) of 100 mM choline chloride. The choline response of the wildtype BetI and the initial library is given in (a) and (b), while those of the selected pools are shown in (c)/(d) (Run-1) and in (e)/(f) (Run-2). This library pool was aliquoted into six batches, each of which was individually subjected to the OFF/ON selections28 in media containing different concentrations of choline chloride. To obtain choline-inducible switches (Run-1), we conducted OFF-selection in LB medium without exogenously added choline. Briefly, 1 µM of artificial nucleoside dP35 was added to the cell culture, followed by a three-hour shaking incubation (Figure 2A). Flow cytometry analysis revealed that the fluorescent clones were nearly completely eliminated from the OFF-selected population (Figure 2B (c)). It has been shown that dP-kinase activity of hsvTK provides exceptionally efficient and highly selective negative selection with minimal false positives.26,28,36 It should be noted that the majority of this OFF-selected pool remained non-fluorescent even in the presence of choline (100 mM), indicating that most of the survivors were indistinguishable from wildtype (always-OFF). At the same time, however, we observed a minor ‘shoulder’ of the high-signal side of the non-fluorescent populations, which we assumed represents the properly switching variants. To enrich this minor fraction, OFF-selected cells were directly subjected to the ON selection in the presence of 100 mM choline. Here, we added choline into the medium and waited for an hour to allow cells to enter the steady state. We then added kanamycin to select for ON-state switches. It should be noted that kanamycin is more bactericidal than bacteriostatic,37 enabling the completion of ON-selection in a matter of several hours. Because we were planning only one cycle of OFF/ON selection, we allowed the cell culture to shake for three hours. However, the time for selection could be shortened to 1 h

without significantly sacrificing selection efficiency (not shown). After this process, almost all of the survivor pool exhibited choline-induced switching in GFP fluorescence (Figure 2B (d)). We also conducted OFF-selection (dP selection) in the presence of 100 mM choline followed by ON-selection (Kanamycin selection) in the absence of choline (Figure 2A, Run-2) to isolate the choline-repressive switches. Also in this experiment, it appeared that the cell pool was completely dominated by variants with the always-OFF phonotype after the OFFselection (Figure 2B (d)). Nevertheless, subsequent ONselection in the absence of choline drastically changed the population behavior: Almost all of the cells were nonfluorescent in the presence of choline, while almost all of the cells were fluorescent in the absence of choline (Figure 2B (e)). This drastic shift in population phenotype and surprising homogeneity in phenotype of the selection process again indicates the robustness and high fidelity of our selection system. Taking advantage of the fact that the entire selection procedure can be completed only by liquid handling, we also conducted selection experiments for Bet-ON switches and for Bet-OFF switches, in which the choline concentration of the ‘high’ state was set to 10 mM (Run-3 and Run-4, respectively). Here again, we observed the drastic changes in the population behavior of the OFF-selected and then ON-selected pool in both runs (Supplementary Figure 1), further confirming the reliability and the power of the selection using hsvTK::APH. We also conducted consecutive OFF/ON selection, in which the concentration of the ‘high’ state was set to 1



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mM (Run-5 and Run-6, respectively). Under these conditions, there were virtually no survivors: Prolonged shaking of the selected culture resulted in the growth of cells with the always-ON phenotype (data not shown). We believe the initial variant pool (2 × 105 in library size) did not contain a single correct clone that met the selection requirement, and some selection escapees arose either by mutational inactivation of the negative selector (hsvTK)36 or by stochastic cell-to-cell variation in cell growth/expression. Characterization of BetI variants. From the final variant pool of both Run-1 (Figure 2) and Run-3 (Supplementary Figure 1), 92 variants were randomly picked and independently scored for their fluorescence in the medium with (100 mM) or without choline (Supplementary Figure 2). Virtually all mutants exhibited high cellular fluorescence in the presence of choline, whereas they did not in its absence. We observed significant variation in the expression level in induced conditions (100 mM choline for Supplementary Figure 2 AB and no choline for Supplementary Figure 2 CD). This variation indicates that the kanamycin selection (ONselection) was cut-off rather than graduated or weighted. This selection allowed the survival of all the clones that could have scored a certain expression level, and little competition, if any,

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among the survivors was taking place. This could be related to the fact that we set the selection condition in such a way that it was decoupled from cell growth.28 Eight mutants with the highest ON/OFF ratio among the 92 variants picked as “Bet-ON switches” (Run-1 in Figure 2 and Run-3 in Supplementary Figure 1) were subjected to further analysis. All of them exhibited graded increases in cellular fluorescence with the concentration of choline added to the media (Figure 3A). We did not see much variation in the maximum expression level in the ON-condition (100 mM choline), but we observed significant variation in the fluorescence signal in the absence of choline (on the level of leakiness) (Figure 3A). In terms of sensitivity, BetON(1-B9) was the best mutant (EC50 value ~1 mM) (Table 1). With the exception of BetON(1-A11) and BetON(1-B9), variants were unique in their genotype (Table 1). Most notable was the mutation I22T – which was shared by the three mutants BetON(1-A3), BetON(1-A6), and BetON(1-H6) – which exhibited the lowest leaky expression (and therefore the highest stringency) and achieved a 130-150-fold induction. This mutation was found in our previous, colony-based fluorescence screening29 for the BetI mutants that enabled tight control of the T7P-based system for protein overexpression.

Figure 3. Dose-responsive expression of T5 promoter fused with betO in the cell expressing (A) Bet-ON and (B) Bet-OFF variants. Relative fluorescence per cell is plotted as a function of choline chloride concentration.

We also randomly chose 94 reversely responsive BetI mutants from Run-2 (Figure 2) and Run-4 (Supplementary Figure 1), from which eight mutants with the highest ON/OFF ratio were analyzed. In this instance, we observed variation in particular in the maximum expression level rather than the level of stringency (Figure 3B). In terms of the level of leaky expression in the OFF-state condition (with 100 mM choline), they were approximately 10 times higher than those for choline-inducible (Bet-ON) mutants with the lowest leakiness. Thus, choline-repressive mutants (“Bet-OFF mutants”) have apparent room for improvement in terms of their stringency. Interestingly, none of the eight sequenced Bet-OFF variants possessed the four mutations (A84E, L88F, L183P, and L192P) previously shown to alone confer the reversed phenotype to BetI.29 Five variants shared the same mutation S135P (Table 1), which had not been found among BetI variants that enable choline-repressible T7 promoters.29 The Mutant BetOFF(2-B4) had only one synonymous mutation R167P, indicating that this mutation alone conferred the reversed phenotype to BetI. Mutants BetOFF(2-G3) and BetOFF(2-C6) had

two mutations each (Q119R/L134P and R68C/V138L, respectively). Mapped on the modeled structures, they were rather remote to the previous ones (Supplementary Figure 3). We do not know exactly why we found a completely new set of mutations that confer a reversed phenotype to BetI as opposed to re-discovering the previously found mutations. It may be that the previous reversing mutations were discovered in a completely different context (they were obtained from BetI variants GFP-screened for choline-induced repression of the T7 promoter). Thus, the behavior of BetI could be reversed with many different mutations, as has been reported for TetR.38 Mono-dispersed and graded response of BetI mutations to choline concentration. In metabolic engineering and synthetic biology applications, precise and graded control of expression is often preferred. More importantly, the entire cells should respond in a uniform manner at all inducer concentrations.39 However, many of the induction systems, including the most popular ones, such as lactose- and arabinose-promoters, have been known to exhibit all-or-none behavior,40,41 whereby


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intermediate concentrations of inducers give rise to subpopulations of cells that are fully induced and un-induced. This has been ascribed to the autocatalytic topologies of these induction systems. In the ara operon, for instance, genes encoding arabinose importers (araE and araFGH) are under arabinoseinducible control through AraC, thereby making an autocatalytic loop.41 Because the bet operon also has this autocatalytic loop, whereby BetT (the choline transporter) is placed under the control of the Bet promoter and is positively regulated with choline,42 we were originally concerned that Bet-ON/Bet-OFF systems could respond to choline in an all-or-none fashion.

Flow cytometry revealed that both Bet-ON and Bet-OFF switches responded to choline with high homogeneity at all concentrations (Figure 4). To confer population homogeneity on arabinose-induced gene expression, it is necessary to additionally (over-) express transporters to override the positive loop.43,44 The Bet-based systems described herein behaved uniformly without such an engineering step. This ‘portability’ of the mono-dispersed and graded response could be another advantageous feature of the Bet systems, facilitating the fine control of any genes in different strains.

Figure 4. Flow cytometric analysis of the cell with (A) Choline-inducible and (B) Choline-repressive control of GFP expressions. For each of the cell harboring BetII22T (A) and BetIS135P (B), the distribution in the cellular fluorescence was measured in the media with different concentrations of choline chloride.



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Strain-dependence in the switching properties of Betswitches. Because the identity of the components (BetI and betO) is almost completely identical to that of the endogenous components, Bet switches that are constructed on the plasmids inevitably crosstalk with chromosomal counterparts. Given this unique situation, we analyzed the switching behavior (dose response to choline) of Bet-ON/Bet-OFF switches in five different E. coli strains (Supplementary Figure 4). Although qualitative behaviors of Bet-switches were unchanged in all strains, their specifications (EC50, maximum expression level, stringency, and hill coefficient) varied depending on the strains (Table 2). The choline-induced system (Bet-ON systems) performed better in DH10B and MG1655, exhibiting a dynamic range of the expression level that was ten times higher, mainly due to the high stringency in the OFF-state condition (without choline). Although BL21-AITM and XL1 Blue were superior in their sensitivity to the inducer (choline), they seemed not to be good choices for achieving highly stringent expression control using BetII22T. Tighter control of gene expression in these strains can probably be easily achieved by conducting OFFselection in these very strains. The same holds true for cholinerepressive systems (Bet-OFF systems): We observed small but significant variation in specifications of BetIS135P, whereas its qualitative behavior remained the same in all tested strains. The complete removal of the chromosomal bet operon from MG1655 resulted in a significant loss of stringency in Bet switches and elevated the overall signal in all choline concentrations. We assume that removal of bet operons, including genes encoding choline-metabolizing enzymes (betAB), elevated the effective cellular concentration of choline. In agreement with this assumption, the removal of the bet operon also elevated the apparent sensitivity of Bet switches to choline (Table 2).

Bet-switches as pathway controllers. Graded and singly dispersed transcription control (Figure 4) is a preferred feature in metabolic pathway engineering. Encouraged by this preference, we tried to convert constitutive carotenoid operons into

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choline-inducible ones. We took a part of the natural carotenoid gene cluster containing crtE, crtI, and crtB (a total of 4,874 bases) from Pantoea agglomerans.45 This sequence has been repeatedly used for bacterial lycopene production46 and for color screening for improved isoprenoid pathways.47 This DNA fragment was subcloned into the vector pBR322. Although there are no annotated promoters on the 122 bases upstream of crtE, we observed the constitutive production of lycopene of E. coli harboring the plasmid (data not shown). The analysis of the DNA sequence of 1,063 bp upstream from the CrtE-encoding region of the resultant plasmid – using the program Neural Network Promoter Prediction48 and automated genome annotation tool (BPROM)49 – identified seven and two putative promoter sequences (Supplementary Figure 5). None of them were on the 122-bp sequence that originated from P. agglomerans. Thus, we did not know exactly where the transcription of the lycopene biosynthetic genes starts. Given this, we decided to insert the betO sequence into the upstream neighboring region of the reading frame of CrtE, the first committing enzyme of the lycopene pathway (Figure 5A). The resultant plasmid was transformed into E. coli harboring choline-inducible mutant (I22T) of BetI (Figure 5B). The lycopene production of the transformant cells was found to increase progressively with the increase in the concentration of choline in the medium (Figure 5C). In contrast, with the coexpression of the choline-repressive mutant (S135P) of BetI, the cell harboring this operon exhibited increasing pigmentation and carotenoid accumulation with the decreasing concentration of choline (Figure 5C). Thus, by minimal modification, the heterologous, constitutively expressing operon could be quickly converted to the choline-controlled pathway. With this prototype constructed with minimal modification, we could not completely turn off lycopene biosynthesis at the OFF-state condition (repressive culture condition). Notably, choline-controlled repression of crtEIB must block not only the promoter landing on the betO-flanking putative promoter via steric hindrance/competitive binding but also the elongation complexes coming from the (putative) promoters located further upstream (which is known to be far more inefficient 50).


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Figure 5. Conversion of natural constitutive lycopene producing operon into choline-inducible/-repressive ones. (A) Strategy to regulate metabolic flux to the lycopene pathway by switching the transcription of crtE. (B) Establishment of E. coli strain capable of choline-responsive lycopene production. (C) Pigment accumulation of Cells harboring choline-inducible (left) and cholinerepressive (right) operon by changing choline concentration in the media. Each bar represents the average of three independent experiments, while error bars represent the standard deviation. Two-input Boolean logic gates consisting of synthetic choline/tetracycline promoters. Bacterial promoters can be combined with multiple operator sites, enabling the establishment of complex gene regulations with multiple inputs. To rapidly and reliably upgrade T5P/betO into two-input gates, we added another operator tetO in the 25-nucleotide region between the 35 and -10 boxes.51,52 The expression of the resultant T5P::tetO/betO should be negatively regulated both by BetI and the tetracycline-responsive repressor TetR. In addition, the regulator plasmid was also modified so that it additionally expressed TetR constitutively, yielding pAC-PL-betI-tetR. E. coli strain MG1655 was co-transformed with the two plasmids and was subjected to fluorescence analysis in the presence/absence of choline/anhydrous tetracycline (aTc) (Figure 6A). In the absence of inducers, the cell exhibited virtually no fluorescence, due to the repression both by BetI and TetR. The cellular fluorescence remained low when either choline or aTc was fed alone. By feeding both inducers, we observed GFP fluorescence. This is a typical behavior as AND-gate, indicating both the repressors BetI and TetR tightly and independently regulates the genes downstream from the T5P::tetO/betO promoter.

To be successfully integrated into the complex synthetic networks as designed, each synthetic sub-network should behave predictably in multiple contexts. We replaced cholineinducible BetI (BetII22T) with reverse-type BetI (BetIS135P) of the regulator plasmid and examined the behavior of the T5P::tetO/betO promoter upon co-existence of the resulting plasmid. On this occasion, the repression by BetI was cancelled by NOT feeding choline, whereas the repression by TetR was cancelled by feeding aTc. With this construct, GFP fluorescence was observed only in the (choline/aTc = 0/1) condition (Figure 6B). This is typical behavior of the so-called “aTc-NIMPLY-choline” gate. The same applies for reversing the function of TetR.38 Replacement of TetR(wt) with TetRA56P, I57L, E58V resulted in the construct that produced GFP expression only in the (choline/aTc = 0/1) condition, behaving as the “choline-NIMPLY-aTc” gate (Figure 6C). Finally, we constructed and tested the regulator plasmid expressing BetIS135P and TetRA56P, I57L, E58V. As was expected, GFP under the control of the T5P::tetO/betO promoter was expressed only in the medium containing neither of the inducers (NOR gate) (Figure 6D).



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Figure 6. Two-input gates controlled by choline and anhydrous tetracycline (aTc). (A) Choline-aTc AND gate. (B) aTcNIMPLY-choline gate. (C) Choline-NIMPLY-aTc gate. (D) Choline-aTc NAND-gate. The T5 promoter was fused with tetO (core region between -35/-10 boxes) and with betO (proximal region right downstream the -10 box). The input/output function was measured by GFP fluorescence in four different conditions. Each bar represents the average of three independent experiments, while error bars represent the standard deviation. Inlets indicate the appearance of the pellet of the cell harboring corresponding plasmids. BetI-ON: BetII22T, BetI-OFF: BetIS135P, TetR-ON: TetR(wt), TetR-OFF: TetRA56P, I57L, E58V. CONCLUSIONS: Even very simple synthetic networks rarely behave as designed from the beginning because each of the network components that have been chosen lack the specific quantitative properties required in their individual context.21,53,54 Network functions, especially those made of many parts and/or layers, come into existence based on a finely balanced interaction, and their behavior is inherently affected by small changes in the specification of each component. Thus, expansion of the parts lineup and addressing the wide range of specifications (operator binding affinity and specificity, dynamic ranges, cellular half-life, etc.) are highly desirable for improving the degree of freedom for network designers.6,7,55,56 In particular, inducible switches are important because they can be regarded as network components whose specifications can be exogenously adjusted after construction. The present work provided a number of choline-adjustable transcription controlling systems with different specifications. Additionally, we demonstrated that the net repression strength could be finely and continuously adjusted by changing the

choline concentration, without the appearance of all-or-none behavior. Unlike the T7-based Bet switches that we previously reported,29 the set of genetic switches presented in this work were evolved not for overexpression (whose induction is highly lethal) but for the establishment of the compact switching systems (without the need for additional polymerases) that are useful for the tight but graded control of the expression of genes of interest (Figures 1, 2, and 4). The resultant switches were shown to meet the requirements of the plug-and-play applications for prolonged control of biosynthetic pathways (Figure 5) and, in addition, acted a component in two-input Boolean logic gates (Figure 6). The selection method used in this work is advantageous in that both ON- and OFF-selection can be completed in liquid media, thereby allowing multiple runs of the selection process in parallel (Figure 2 and reference28). By decoupling selection from growth, both ON- and OFF-selection could be completed within a few hours,28 although we allowed 3 hours of incubation time for each selection process. By a single round of rapid, seamless, and parallel operation of OFF/ON selection, we

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could isolate numbers of properly functioning BetI-variants (Figure 2 and Supplementary Figure 3) with negligible contamination of false positives. Given the dramatic improvement in liquid-based selection platforms,22,26-28,57 a build-then-mutate approach58 may be a routine and standardized approach for the fast-track construction of genetic switches and circuits.

MATERIALS AND METHODS: Bacterial Strains, Media, and Chemicals. E. coli strain MG1655 was used throughout this work, and XL10 Gold (tetr) (Stratagene, La Jolla, CA) was used for cloning purposes. E. coli strains were incubated in LB medium (2.0% (w/v) LB; Invitrogen) or on LB-agar plates (2.0% (w/v) LB, 1.5% (w/v) agar; Nacalai Tesque, Kyoto, Japan) at 37˚C. Antibiotics were used at the following concentrations: 30 µg/ml chloramphenicol (Cm; Nacalai Tesque), 100 µg/ml ampicillin (Amp; SigmaAldrich, St. Louis, MO) , and 100 µg/ml kanamycin (Km; Sigma-Aldrich, St. Louis, MO, USA). Stock solution (1 M) of choline chloride was prepared by dissolving appropriate amounts of the compound in LB medium (Invitrogen) and filter sterilized through a 0.2 µm cellulose acetate filter (MN Sterilizer CA, Macherey-Nagel GmbH & Co. KG, Düren, Germany), and stored at 4°C. Anhydrotetracycline (aTc) stock solution (100 µg/ml) was prepared by dissolving 2 mg/ml aTc (Clontech) 50 µl in H2O 500 µl and EtOH 450 µl and stored at -20°C. Plasmid Construction. All plasmids used in this study are shown in Supplementary Table 1. pET23d-pT5/betO-sfgfphsvtk::aph was constructed as follows. The T7 promoter of pET23d-pT7/betO-sfgfp29 was replaced with the T5 promoter. The open reading frame of the dual selector HSVTK::APH28 was PCR-amplified with an appropriate RBS sequence and subcloned into the resulting plasmid. pET23d-pT5:tetO/betOsfgfp was constructed as follows. Tet operator (tetO: 5’TCCCTATCAGTGATAGAGA-3’) was inserted between -35 and -10 of the T5 promoter by using the FASTR cloning method59 using the following primers (forward primer: 5’TTT TGC TCT TCA TT AAT TGA ACG TTC AAT TAA CCC TCT AGA AAT TAA TTT G-3’, reverse primer: 5’TTT TGC TCT TCA TAA CCT ATT ATA TCT CTA TCA CTG ATA GGG AAG CAA ATA ATT TCG CGG GAT CGA G-3’: bold and underlined are LguI restriction sites and annealing sites, respectively). For the plasmid map and nucleotide sequence, see Supplementary Figure 6. pBR-betO-LYC was constructed by PCR to amplify the part of natural crt operon from Pantoea agglomerans from pACLYC45 and subcloned into the HindIII/SalI site of the pBR322 by using the SLIC cloning method.60 Next, bet operator sequence (5’- TTA ATT GAA CGT TCA ATT AA -3’) was inserted upstream of the crtE gene in the resulting plasmid using ExSite-PCR61 using the following primers (forward primer: 5’- TTT TTC TAG ATT AAT TGA ACG TTC AAT TAA CAA TTC AGC GGG TAA CCT TGC -3’, reverse primer: 5’- TTT TTC TAG ATC TTT TAG TAT CAG TTA ACC GTA GAC CGC -3’: bold and underlined are XbaI restriction sites and annealing sites, respectively). ON/OFF selection of T5-based BetON/OFF switches. The library plasmid pAC-PL-[betI]lib (plasmid map and nucleotide

sequence provided in Supplementary Figure 7), which was prepared by random mutagenesis of the entire betI using error prone PCR (library size ~ 1 × 105),29 was transformed into MG1655 harboring pET23d-pT5/betO-sfgfp-hsvtk::aph. A portion (1/100,000) of the transformant was plated on LB plates to determine the selection size (5.2 × 107: sufficiently larger than the original library size 1 × 105). The transformants were subjected to ON/OFF selection as previously described.28 Shortly after, overnight cell cultures with or without 100 mM choline chloride were diluted by 100-fold into fresh LB-CarbCm medium containing 0-100 mM choline chloride. After shaking at 37°C for 1 h, dP (final concentration 1 µM) was added to these cultures, followed by shaking at 37°C for 3 h (OFF selection). The resultant cultures (ON-selected pools) were divided into two pools: one was stored as an intermediate pool for analysis, whereas the other pools were rinsed twice with saline and re-suspended with a 100-fold volume of fresh LB-Carb-Cm medium containing 100 or 0 mM choline chloride. After shaking at 37°C for 1 h, Km (final concentration 100 µg/ml) was added to these cultures, followed by shaking at 37°C for 3 h (ON selection). These cultures were rinsed with saline and diluted into fresh LB medium and then shaken at 37°C for 3 h for cell growth. The resultant cultures (ON/OFFselected pools) were stored for analysis. Fluorescence analysis. A 5-µl aliquot of a pre-culture was inoculated into fresh LB medium (500 µl in 96-deep-well plates with a V-shaped bottom (Greiner Bio-One, Frickenhausen, Germany)) containing 0 - 100 mM choline chloride and the appropriate antibiotic. The mixture was then shaken for 12 h at 37°C. Cell cultures were subsequently transferred into 96-well microtiter-plates with clear bottoms (Nunc 269620, Thermo Scientific, Waltham, MA, USA). The fluorescence intensity (488 nm excitation, 507 nm emission) of each sample was measured using a fluorescence microplate reader (Fluoroskan Ascent, Thermo Scientific) with excitation at 485 nm and emission at 510 nm. The fluorescence of the cell suspension was normalized to the optical density, which was measured using the microtiter-plate reader (SPECTRAmax, Molecular Devices, Sunnyvale, CA, USA) at 600 nm. The fluorescence output of the strain harboring pACmod and PL-betI was also measured to determine the background fluorescence. All of the reported fluorescence values were obtained by subtracting this background fluorescence from the actual sample values. Flow cytometric analyses of the cell mixtures were conducted as follows: after recovery from the glycerol stock, the cell mixture was grown in LB medium in test tubes (at 37°C at 200 rpm). A 20-µl aliquot of an overnight pre-culture was inoculated into fresh LB medium (2 ml in 48-deep-well plates with a V-shaped bottom) containing 0 or 100 mM choline chloride and the appropriate antibiotics. Approximately 100,000 cells were applied to a MACS Quant VYB (Miltenyi Biotech, Bergisch-Gladbach, Germany) equipped with a 488 nm laser and appropriate filter sets for sfgfp (525/50). The data were analyzed using the MACSQuant Analyzer (Miltenyi Biotech, Bergisch-Gladbach, Germany). Carotenoid pigment analysis. The carotenoid production level was analyzed as previously described62 with a slight modification. Plasmids (pAC-PL-betI, pBR-betO-LYC) were

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transformed into MG1655 cells, and the transformants were plated onto LB (Amp/Cm) agar plates to form colonies. These colonies were picked and inoculated into 500 µl of LB (Carb/Cm) medium in a 48 deep-well plate and cultured at 37°C, 1000 rpm, for 14 h. An aliquot (20 µl) of these precultures was transferred to 2 ml of LB (Carb/Cm) in a test tube and cultured at 37°C, 200 rpm, for 14 h. The cells were harvested, washed with saline, and centrifuged to obtain cell pellets, and the supernatants were discarded. After briefly vortexing the cell pellets, 1 ml of acetone was added to each of the pellets, and they were immediately vortexed for another 10 min to extract the carotenoids; this was followed by centrifugation. The absorbance spectra (350–650 nm at 5-nm intervals) were analyzed for each of the acetone extracts by using a SpectraMax Plus Absorbance Microplate Reader (Molecular Devices, Sunnyvale, CA). The pigmentation level of each culture was determined from the lambda max of the resulting extract by using the molar adsorption coefficients of the lycopene (475 nm, 185,000 M−1cm−1).

ASSOCIATED CONTENT Supporting Information Other sets of OFF-/ON-selections to isolate cholineinducible (Run-3) and choline-repressive (Run-4) transcriptional switches, switching properties of single-mutant BetIs, structural mapping of reversing mutations of BetI, straindependence in the switching properties of Bet-switches, and promoter prediction analysis of the 5'-UTR region upstream of crtEIB operon. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *Tel/Fax: +81-43-290-3413 E-mail: [email protected] (DU) Notes The authors declare that they have no competing interests. Author Contributions All authors were involved in the experimental design. KS and MT performed experiments. KS, MT, SK-N, and DU analyzed the data. SK-N, KS, and DU supervised the project. KS, MT and DU wrote the manuscript. Funding Sources This work was partially supported by the Commission for the Development of Artificial Gene Synthesis Technology for Creating Innovative Biomaterial from the Ministry of Economy, Trade and Industry (METI), Japan, and the Precursory Research for Embryonic Science and Technology (PRESTO)

Page 10 of 20

program of the Japan Science and Technology Agency (JST). Funding for open access charge: METI.

ACKNOWLEDGMENTS We would like to thank to Dr. Yoichi Mashimo (Dept. Medicine, Chiba University) for consultations on the se1quence analysis. We also thank Dr. Maiko Furubayashi (Massachusetts Institute of Technology) for thoughtful comments on this manuscript.

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Table 1. Mutations found in the BetI variants and their specifications. Amino acid substitution (nucleInduction Variants otide substitution in parenthesis) fold [-]

Repression fold [-]

EC50 [mM]

Hill coefficient

Bet-ON (1-A3)

I22T(T65C), Q100Q(G300A)

288±153

-

6.3±1.4

1.3±0.03

Bet-ON (1-A6)

I22T(T65C), M106T(T317C), A108A(C324A)

313±23

-

13.9±0.3

1.3±0.03

Bet-ON (1-C2)

S8P(T22C), Y123D(T367G)

33±1

-

2±0.1

1.2±0.05

Bet-ON (1-H6)

I22T(T65C)

353±170

-

9.6±0.1

1.3±0.04

Bet-ON (3-A11)

I35T(T104C)

22±4

-

2.3±0.3

1.2±0.01

Bet-ON (3-B9)

I35T(T104C)

19±4

-

2.1±0.5

1.2±0.1

27±5

-

2.6±0.3

1.2±0.1

26±6

-

2.4±0.1

1.2±0.1

Bet-ON (3-C10) Bet-ON (3-D8)

I35T(T104C), L145L(G435A), D195G(A584G) Q13R(A38G), D52G(A155G), M121R(T362G)

Bet-OFF (2-B3)

S135P(T403C)

-

35±2

2.1±0.02

1±0.01

Bet-OFF (2-B4)

R167P(G500C)

-

17±1

7.6±1.6

1.6±0.4

Bet-OFF (2-C6)

R68C(C205T), V138L(G412T)

-

20±3

1.3±0.3

1.1±0.2

Bet-OFF (2-D4)

S135P(T403C)

-

34±3

3.2±0.2

1.1±0.02

Bet-OFF (2-D6)

S135P(T403C), T194A(A580G)

-

36±4

1.9±0.1

1±0.1

Bet-OFF (2-E1)

R38H(G113A), S135P(T403C)

-

32±5

2±0.1

1.5±0.1

Bet-OFF (2-E4)

R75R(A225G), S135P(T403C)

-

30±2

3.3±0.5

1.1±0.1

-

49±8

0.5±0.1.

1.3±0.1

8±3

-

25.7±3.2

1.6±0.2

Bet-OFF (2-G3) wildtype

Q119R(C355G), L134P(T401C) -

Values in the table represent the average of three independent experiments, with standard deviations. For all samples, R2 values was higher than 0.98 for all variants, while that for wildtype BetI was 0.95.

Table 2. Behavior of Choline-inducible and Choline-repressible expression controllers in various strains. BetII22T BetIS135P Induction Hill CoeffiRepression EC50 [mM] EC50 [mM] fold [-] cient [-] Strain fold [-]

Hill Coefficient [-]

MG1655

6.5±1

159±6

1.4±0.05

1.6±0.2

18±2.9

1.4±0.03

MG1655∆bet

5.4±0.6

18±2

1.6±0.2

0.8±0.2

23±4

1.1±0.1

DH10B

14±0.2

317±165

1.5±0.02

2.6±0.2

52±4

1.3±0.04

BL21-AITM

1.2±0.06

15±0.6

1.1±0.1

0.8±0.04

34±9

1.1±0.05

XL1Blue

1.8±0.1

11±0.4

1.2±0.02

0.3±0.03

18±1.5

1.4±0.05

Values in the table represent the average of three independent experiments, with standard deviations. For all samples, R2 values was higher than 0.97.

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Figure 3 155x60mm (150 x 150 DPI)



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Figure 4 144x64mm (150 x 150 DPI)


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Figure 5 89x77mm (300 x 300 DPI)



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Figure 6 99x131mm (150 x 150 DPI)


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