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Construct Functional Feedforward Loop Biological Circuits in a Cell-Free System and in Cells Shaobin Guo, and Richard M. Murray ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.8b00493 • Publication Date (Web): 21 Feb 2019 Downloaded from http://pubs.acs.org on February 22, 2019

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ACS Synthetic Biology

Construct Functional Feedforward Loop Biological Circuits in a Cell-Free System and in Cells Shaobin Guo†,*, Richard M. Murray†,§ †

Department of Biochemistry and Molecular Biophysics, California Institute of Technology, Pasadena, California United States

§

Department of Control and Dynamical Systems, California Institute of Technology, Pasadena, California United States

*

Corresponding Author: [email protected]

Abstract: Cells utilize transcription regulation networks to respond to environmental signals. Network motifs, such as feedforward loops, play important roles in these regulatory networks. In this work, we construct two different functional and modular incoherent type 1 feedforward loop biological circuits in a cell-free transcription-translation system and cells. With the help of mathematical modeling and the cell-free system, we are able to streamline the design-build-test cycles of the circuits, in which we characterize and optimize these circuits in vitro to confirm that they function as expected before implementing them in vivo. We show that performances of these circuits from in vitro studies closely recapitulate those from in vivo experiments. We demonstrate that these feedforward loops show dynamic response and pulse-like behavior both in vitro and in vivo. These novel feedforward loop network motifs can be incorporated in more complicated biological circuits as detectors or responders. Keywords: cell-free system; feedforward loop; biological circuit; prototyping; synthetic biology. Transcriptional regulation networks are important components of the cell regulation system as cells use them to adapt to environmental changes.1 They contain a small set of recurring interaction patterns called network motifs.2 Different network motifs use distinct regulation mechanisms to respond to signals; as one of the essential mechanisms, feedforward control is widely used by bacteria and yeasts to respond to environmental signals.3, 4 The network motif involving feedforward control is called a feedforward loop (FFL). The FFL consists of three components: 𝑥, 𝑦, and 𝑧. Transcription factor 𝑥 is a regulator for both 𝑦 and 𝑧, and transcription factor 𝑦 is also a regulator for 𝑧. There are eight different subtypes of FFLs in total.3 Among all the FFLs, incoherent type 1 FFL, in which 𝑥 activates both 𝑦 and 𝑧 but 𝑦 represses 𝑧, has been shown to be a pulse generator as a result of a time delay between the activation of 𝑧 by 𝑥 and the repression of 𝑧 by 𝑦 (Figure 1A). Incoherent type 1 FFL also shortens the time needed for the production of 𝑧 to reach half of the steady state level; hence a response accelerator. This type of FFL is used by E. coli to execute rapid adjustments to environmental changes, such as glucose starvation.5 As synthetic biology progresses, we will need more independent, modular and functional network motifs, such as FFLs, as building blocks to construct more complex transcription 1

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regulation networks with unique functions in cells or artificial cells.6-9 Currently, there are limited FFLs that can be used for this kind of purpose. To that end, in this work, we design, test and build two different novel incoherent type 1 FFL biocircuits in vitro and in vivo. We use an Escherichia coli-based cell-free transcription-translation system (TX-TL), which is optimized for biocircuits, as a testbed for prototyping the FFLs in vitro.10-13 By using TX-TL, we are able to significantly shorten the time spent on tuning the FFL biocircuits and streamline the process of optimization, even for biocircuits with dynamic behavior. Then we implement the optimized FFLs in E. coli and verify the functionality of the FFLs in vivo. We show that experimental results of the FFLs that we get in TX-TL closely recapitulate those in cells, suggesting that it is worth prototyping biocircuits in TX-TL before testing them in cells. The two novel FFL biocircuits that we build can be used as functional modules in more complex transcription regulation networks both in vitro and in vivo. Results and Discussion As mentioned above, the FFL circuit is made of three components 𝑥, 𝑦, and 𝑧. In order to control the exact starting point of the FFL dynamics, we add another component (𝑢) to the FFL circuit for an additional level of control (Figure 1B). In this case, only complex 𝑥: 𝑢 can initiate the dynamics of the FFL by activating both 𝑦 and 𝑧. And the activation of 𝑦 and 𝑧 is positively correlated with 𝑢, so 𝑢 can be seen as an inducer that is required for 𝑥 to function. After examining the design, we searched for biological parts that fit the FFL circuit requirements. For the first trial, we decided to use the AraC-arabinose activation complex as 𝑥: 𝑢, a transcription factor TetR as the repressor 𝑦 and deGFP fluorescent protein with a corresponding combinatorial promoter as the output 𝑧 (Figure 1C).14-16 The transcription factor araC, controlled by a constitutive promoter J23151,17 binds to the promoter pBAD and activates the transcription of downstream genes (tetR and degfp) only in the presence of the inducer arabinose. On the other hand, the transcription factor TetR binds to the operator site tetO and represses the transcription of the degfp gene, regardless of the presence of AraC:arabinose. Previous work has shown that in a combinatorial promoter where the tetO site is downstream of the pBAD site, the repression imposed by TetR protein always dominates the activation.18 Because of the time delay between activation by AraC:arabinose and repression by TetR, the degfp gene will initially be transcribed, and then the transcription will be repressed after TetR proteins accumulate to a certain threshold concentration. At the same time, the LAA-ssrA degradation tag on the deGFP protein leads to the degradation of deGFP by ClpX protein.19 As a result, the green fluorescence signal of deGFP protein, which can be measured, will initially increase and then decrease, generating a pulse-like behavior, like the simulation shown in Figure 1D. Simulations of this FFL circuit were performed using a TX-TL toolbox developed in the lab.20 Also in Figure 1D, we simulated how the varied concentrations of the repressor TetR DNA can affect the deGFP dynamics with all the other components as constants. As the simulation result shows here, increasing TetR DNA concentration not only brings down the peak deGFP concentrations but also shifts the peak to the left, suggesting that it takes less time for TetR protein to accumulate to the threshold level when there is more TetR DNA to start with. 2


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Figure 1: A: An illustration of an incoherent type 1 FFL. Arrows mean activation and bars mean repression. B: An illustration of aFFL with an additional input. C: A FFL circuit design based on AraC-TetR-deGFP. In this illustration, dark blue curved block arrows are promoters, yellow half circles are ribosome binding sites (RBSs), gray straight block arrows are coding sequences (CDSs), and orange T-shape block are terminators. D: A simulated time course of a FFL using the TX-TL toolbox. Three curves are deGFP protein concentrations over time with varied initial TetR DNA concentrations at 1nM, 5nM, and 10nM. Initial concentrations for AraC DNA and deGFP DNA are 10 nM and the arabinose concentration is 0.2%. E: Experimental results of the FFL in TX-TL with 10 nM AraC linear DNA, 10 nM deGFPssrA linear DNA, 10 nM ClpX linear DNA, 0.2% arabinose, and varied TetR linear DNA concentrations. F: The time course results of the FFL in cells. The deGFPssrA protein is tagged with the LAA-ssrA degradation tag and deGFP does not have a degradation tag. G: The time course results of the FFL in cells induced with a range of arabinose concentrations. For both F and G, data were average from three independent repeated wells and then were normalized using OD600 readings to get the fluorescence reading for each cell (normalized fluorescence unit, NFU). Error bars represent one standard deviation from three independent experiments.

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The Escherichia coli-based cell-free transcription-translation system (TX-TL) is a cell-free system based on S30 cell extracts, which has been optimized for in vitro biocircuits testing.13 Unlike traditional cell-based tests, TX-TL does not require cell growth and it can start transcription and translation as soon as DNA is added. Because of the simplicity and efficiency that TX-TL system can provide in biocircuits prototyping, we decided to use the TX-TL for our initial tests of the FFL. We constructed the FFL shown in Figure 1C using Golden Gate assembly, in which we mixed all the different parts in one-pot mixtures.21 Then we used these mixtures as a template to amplify the corresponding linear DNAs containing Promoter-RBSCDS-Terminator via PCR. Previous work has shown that, besides plasmids, linear DNAs can also be used in TX-TL for fast circuit prototyping with the protection from the RecBCD inhibitor bacteriophage gamS protein.10 For each construct (pCon-AraC, pBAD-TetR, and pBAD-tetOdeGFPssrA), we varied the promoters until we found the best candidates to test in cells (see Supplementary Materials Figure S1 for the promoter screening). TX-TL experiments were run by mixing TX-TL extracts and TX-TL buffers with linear DNAs (AraC, TetR, and deGFP) and the inducer arabinose in a multi-well microplate at 29°C. Figure 1E showed the experimental results from the best version of AraC-TetR-deGFP FFL biocircuit in TX-TL (see Supplementary Materials Figure S2 for other candidates). As we can see, experimental data closely recapitulated the simulation results and the FFL circuit showed pulselike behavior at all conditions tested. As a higher concentration of TetR DNA was used in TXTL reaction, the peak GFP signal was lower and the time that it took for GFP signal to reach the peak was shorter; this is also consistent with our observations in the simulation. We also noticed that the steady state GFP protein concentrations for different starting TetR DNA concentrations were different. This is because TX-TL reactions have limited resources, including RNA polymerases, NTPs, ribosomes, and amino acids.22 The resources required for ClpX to unfold ssrA-tagged deGFP proteins run out gradually, and the resources get used up faster when there are more linear DNAs in the reaction. As a consequence, the GFP signals showed different steady state levels. We then constructed a circular plasmid form of the best version of AraC-TetR-deGFP FFL and transformed them into E. coli cells (see Supplementary Materials Figure S2 for other candidates). In order to distinguish the reduction of GFP signal by ClpX from that by cell division, we made a negative control circuit, in which the deGFP protein was not tagged with the ssrA degradation tag. Figure 1F showed the experimental results of the FFL circuit (where the deGFP is tagged with a degradation tag) and a control circuit (where the deGFP is not tagged) in cells. The dynamics of the FFL circuit in cells were consistent with those in TX-TL experiments, meaning that the FFL biocircuit prototyped in TX-TL indeed showed the same behavior in vivo. In contrast, the control circuit did not exhibit a pulse-like behavior, suggesting that the drop of GFP signal that we saw in the FFL circuit was not a result of dilution but the degradation of GFP proteins by ClpX proteins. We also tested the FFL with titrations of arabinose (Figure 1G) and pulses can be seen in all concentration range. The size of the pulse is positively correlated with the concentrations of arabinose as more deGFP proteins are produced at higher arabinose concentration. 4


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Figure 2: A: A FFL circuit design based on LasR-TetR-deGFP. B: Time course data of the optimized LasR FFL circuit in MG1655 cells. Three AHL concentrations were used in the experiment. GFP data was subtracted by the background and then normalized with OD data to get the normalized fluorescence unit (NFU). Error bars represent one standard deviation from three independent experiments. C: Time course data of another LasR FFL circuit in MG1655 cells. Five AHL concentrations were used in the experiment. 20 ng/mL of aTc was used in all samples. GFP data was subtracted by the background and then normalized with OD data to get the normalized fluorescence unit (NFU). D: Microscope movie snapshots of the cells containing the optimized LasR FFL circuit in a microfluidic device. Each frame was taken 10 minutes apart.

After we successfully built the AraC-TetR-deGFP FFL biocircuit, we chose different components to design and build another novel FFL consisting of a lasR gene (as 𝑥), an inducer N-(3-Oxododecanoyl)-L-homoserine lactone (AHL as 𝑢), a pLas promoter, and a pLas-tetO combinatorial promoter (Figure 2A). The LasR protein, in the presence of the inducer AHL, becomes an activator that can turn on both pLas and pLas-tetO promoters. All the other components work like the AraC version FFL circuit that we built previously. Following the same prototyping protocol in TX-TL as mentioned above, we tested and selected the best version of LasR-TetR-deGFP FFL and transformed the plasmids in cells (see Supplementary Materials Figure S3-4). The experimental results of this FFL circuit in cells (Figure 2B) showed that it generated pulses as expected. When there was no AHL added, we had no response from the circuit. Only when we had significant activation caused by AHL (more than 50 nM in this case) would we see pulses from the circuit in cells, and the highest peaks of the pulses were positively correlated with the inducer concentrations. A second version of the LasR-TetR-deGFP FFL was also tested in cells and the results were shown in Figure 2C; in this version, anhydrotetracycline (aTc) was required to generate significant pulses. But the reaction time of this FFL was faster compared to that of the best version. Depending on the application, we can choose between these two versions of FFLs.

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Not only did we test the best version of the LasR FFL circuit quantitatively in bulk experiments, but we also examined the circuit behavior in a single-cell setup using a microfluidic device, also known as the mother machine.23 The mother machine consists of a series of growth channels that can trap single bacterial cells inside, and is designed to allow growth medium to pass through the channels at a constant flow rate, which results in diffusion of fresh medium into the growth channels as well as removal of extra cells as they are pushed out of the channels into the main trench. As we can see in Figure 2D, while cells were growing and dividing in a narrow comblike channel, some cells showed green fluorescence intensity starting from weak to strong and then back to weak with a constant flow of fresh media containing 50 nM AHL at all time, thus a pulse of GFP signal. This single-cell experiment further demonstrated that the LasR FFL circuit worked in cells. In summary, here we reported two novel incoherent type 1 feedforward loops with different activation inputs and we demonstrated that they can be implemented both in vitro and in vivo. As researchers characterize and engineer more basic parts to use in synthetic biocircuits,24 we can design more biocircuits as building blocks for constructing novel and complex transcription regulation networks in cells or artificial cells. Besides, this work, together with other publications,10-13, 25, 26 serves as demonstrations of the utility of the cell-free TX-TL system in biocircuit prototyping. Methods Cell-free experiment preparation and execution Preparation of the cell-free TX-TL expression system was done according to previously described protocols.27 All TX-TL experiments were run at 29°C. For deGFP, samples were read in a Synergy H1 plate reader (Biotek) using settings for excitation/emission: 485 nm/525 nm, gain 61. Measurements of deGFP fluorescent unit were converted to nM using a purified deGFPHis6 standard to eliminate machine to machine variation (different Bioteks). PCR product preparation and plasmid DNA assembly Linear DNA fragments were amplified using Pfu Phusion Polymerase (New England Biolabs). Fragments were then assembled using Golden Gate assembly. Assembled circular DNAs were transformed into competent cells for amplification: a KL740 strain (lab-made competent strain) if using an OR2-OR1 promoter (29°C) and a JM109 strain (Zymo Research) for all other constructs. Plasmids were prepared using a Qiagen miniprep kit. All the DNA sequences and plasmids used in the work can be found on https://www.addgene.org/Richard_Murray/ and in the Supplementary Materials.

In vivo experiment E. coli strain MG1655 was used for testing FFL circuits in cells. Plasmid combinations were transformed into chemically competent E. coli MG1655 cells, plated on Difco LB+Agar plates 6


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containing selective antibiotics and incubated overnight at 37°C. Plates were taken out of the incubator, and three colonies were picked, separately inoculated into 5 mL of LB containing selective antibiotics, and grown approximately 17 h overnight at 37°C at 200 rpm in a benchtop shaker. These overnight cultures (100 µL) were then added into new tubes containing 5 mL (1:50 dilution) of Minimal M9 casamino acid (M9CA) media containing the selective antibiotics and grown for 4 h at the same condition as the overnight cultures. Then 10 µL cultures were transferred to 96-well glass bottom plate with 290 µL M9 with corresponding experimental conditions. GFP fluorescence (485 nm excitation, 525 nm emission, gain 61), and optical density (OD, 600 nm) were measured using a Biotek Synergy H1 plate reader at 37°C every 5 minutes with shaking (Double orbital, fast, 567cpm) until the end of experiments. Microfluidic device experiment Cells were grown using the same method as in vivo experiments. Then cells were concentrated to OD600 = 10 and loaded into a premade PDMS microfluidic device (mother machine). Then a constant flow of media was pumped into the device and the device was imaged under an Olympus IX81 inverted fluorescence microscope using a Chroma wtGFP filter cube (450/50 BP excitation filter, 480 LP dichroic beam splitter, and 510/50 BP emission filter), with an XFOcitep 120 PC light source at 100 % intensity and a Hamamatsu ORCA-03G camera. Cells were imaged using a 100x phase objective with oil. Corresponding Author *Email: [email protected]. Author Contributions ¶S.G. and R.M.M. conceived the idea. S.G. designed the experiments, performed the experiments and subsequent data analysis, and wrote the manuscript. R.M.M. provided comments on the manuscript. Funding Sources This work was supported by DARPA Living Foundries (Grant number: HR0011-12-C-0065). Notes R.M.M. has ownership in a company that commercializes the cell-free technology utilized in this paper. S.G. claims no competing interest. Acknowledgment We would like to thank Clarmyra Hayes, Zachary Sun, Yutaka Hori, and Vipul Singhal for helpful discussion and suggestions. We would like to thank Murray lab members for useful suggestions.

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