Development of a Bacillus subtilis cell-free transcription-translation system for prototyping regulatory elements

Cell-free transcription-translation systems were originally applied towards in vitro protein production. More recently, synthetic biology is enabling these systems to be used within a systematic design context for prototyping DNA regulatory elements, genetic logic circuits and biosynthetic pathways. The Gram-positive soil bacterium, Bacillus subtilis , is an established model organism of industrial importance. To this end, we developed several B. subtilis -based cell-free systems. Our improved B. subtilis WB800N-based system was capable of producing 0.8 µM GFP, which gave a ~72x fold-improvement when compared with a B. subtilis 168 cell-free system. Our improved system was applied towards the prototyping of a B. subtilis promoter library in which we engineered several promoters, derived from the wild-type P grac ( σ A) promoter, that display a range of comparable in vitro and in vivo transcriptional activities. Additionally, we demonstrate the cell-free characterisation of an inducible expression system, and the activity of a model enzyme - renilla luciferase.


Introduction
Cell-free systems that are based on cellular extracts were originally developed as experimental systems to understand fundamental aspects of molecular biology, cellular biochemistry and for in vitro protein production (Hodgman and Jewett, 2012;Nirenberg, 2004;Sullivan et al., 2016;Zubay, 1973). Synthetic biology approaches are enabling the re-purposing of cell-free systems as coupled in vitro transcriptiontranslation characterisation platforms for the prototyping of DNA based parts, devices and systems (Kelwick et al., 2014). Cell-free transcription-translation systems have been employed to rapidly prototype DNA regulatory elements (Chappell et al., 2013), logic systems (Niederholtmeyer et al., 2015;Shin and Noireaux, 2012;Sun et al., 2014;Takahashi et al., 2015) and medical biosensor devices (Pardee et al., 2014) with workflows that can be completed within several hours. In contrast, typical in vivo approaches may take several days. Another distinguishing advantage of cell-free systems is that they can be coupled with model-guided design strategies to create 'biomolecular-breadboards' that enable the robust cell-free characterisation of bioparts that can then be implemented as final designs in vivo (Siegal-Gaskins et al., 2014). These developments are also enabling cell-free protein synthesis driven metabolic engineering approaches for the biochemical characterisation of novel enzymes and prototyping of biosynthetic pathways (Karim and Jewett, 2016).
Several cell-free systems have been developed, of which, the most well established systems use cellular extracts from Escherichia coli (Garamella et al., 2016), Wheat Germ (Ogawa et al., 2016), Yeast (Gan and Jewett, 2014) or HeLa cells (Gagoski et al., 2016). Additionally, more specialist cell-free systems including the PURE express system, which uses purified cellular machinery rather than cellular extracts, have also been established (Shimizu et al., 2005). Whilst these cell-free systems have been continually improved through developments in the methods used for their preparation (Shrestha et al., 2012) and optimisation of energy buffers Noireaux, 2015a, 2014), there have been fewer reports of Bacillus subtilis cell-free systems. Yet, the development of robust B. subtilis cell-free systems could have applicability to a broad array of microbiology, synthetic biology and industrial biotechnology applications. Applications for B. subtilis are diverse and include the production of industrial or pharmaceutical proteins, and more recently for use as whole-cell biosensors (Harwood, 1992;Pohl et al., 2013;Webb et al., 2016;Westers et al., 2004). Cell-free systems could be applied to support developments across these applications, particularly, where the functionality of the engineered system relates to aspects of the biochemistry, metabolism and/or regulatory processes of B. subtilis as well as potentially other Gram-positive bacteria. For instance, the cell-free prototyping of B. subtilis regulatory elements (e.g. promoter libraries) may provide synergistic benefits when coupled with in vivo studies, such that multiple rounds of cell-free characterisation workflows may result in more rapid iterations of the design cycle towards the final in vivo design (Chappell et al., 2013;Karim and Jewett, 2016;Tuza et al., 2013).
However, the initially reported B. subtilis cell-free systems were typically encumbered by the requirement to use exogenous mRNA, protease inhibitors, DNAse treatments or less efficient energy systems (Legault-Demare and Chambliss, 1974;Leventhal and Chambliss, 1979;Nes and Eklund, 1983;Okamoto et al., 1985;Zaghloul and Doi, 1987) which is perhaps why, despite their potential, these systems have been largely neglected. In the present study, we report on the development and improvement of a B. subtilis cell-free system, using a standardised workflow that has no such limitations. We demonstrate the utility of B. subtilis cell-free transcription-translation systems as a useful tool for genetic regulatory element prototyping through the characterisation of an engineered promoter library that enables a range of comparable in vitro and in vivo transcriptional activities. Additionally, as a step towards additional applications for B. subtilis cell-free systems, we characterise an inducible expression system (a precursor to genetic circuit prototyping) and, characterise the activity of the Renilla (sea pansy) luciferase (a model enzyme).

Strain and plasmid construction
Oligonucleotide primers for plasmid construction and sequencing are listed in Table S2.

GFPmut3b expression vector
The GFPmut3b expression vector pHT01-gfpmut3b was constructed as follows. The insert gfpmut3b was amplified from plasmid pAJW26 (BBa_K316008) using primer pair AJW289/AJW290, the resultant PCR product was purified, digested with enzymes BamHI and XbaI and ligated with the vector pHT01, which had been digested with the same enzymes, resulting in the construction of plasmid pHT01-gfpmut3b (pAJW107). To remove LacI control, lacI was deleted from plasmids pHT01 and pHT01-gfpmut3b as follows: inverted PCR reactions using primer pair AJW320/AJW321 and plasmids pAJW9 and pAJW107 as templates were undertaken, the DNA products were purified, phosphorylated, self-ligated and transformed into E. coli NEB10-beta, resulting in the plasmids pHT01-ΔlacI (pAJW118) and pHT01-ΔlacI-gfpmut3b (pWK-WT). To generate a pHT01-ΔlacI-gfpmut3b construct lacking the −35 and −10 boxes and the region between the two boxes, plasmid pWK-WT was used as the template in an inverted PCR reaction with primers WK5/WK6. The resultant DNA product was purified, phosphorylated, self-ligated and transformed into E. coli NEB10-beta, resulting in the plasmid pHT01-ΔlacI-Δbox-gfpmut3b (pWK-Δbox).

Promoter library construction
To construct the promoter library of clones with changes to the −35 and −10 boxes, inverted PCR was undertaken using pWK-WT as the template and primer pair WK1/WK2. The resultant PCR product was purified, phoshporylated, self-ligated, transformed into E. coli NEB10beta and the colonies cultured on plates incubated at either 30°C or 37°C. This resulted in the production of the pWK(n) plasmid promoter variants. To create targeted changes to the −10 box, inverted PCR was undertaken using pWK-WT and pWK5 as the templates and primer pair WK7/WK8. The products were purified, phosphorylated, selfligated and transformed into E. coli NEB10-beta, resulting in the plasmids pWK403 and pWK501 respectively. Promoter library clones tested in this study are listed in Table S6.

GFPmut3b purification vector
Primer pair RK003 and RK004 were designed to PCR amplify gfpmut3b along with the addition of restriction sites BamHI and HindIII from plasmid pRK1. The subsequent PCR product was designed so that it could be digested with BamHI and HindIII and ligated into pre-digested vector pPROEX HTb to form pRK2a vector in which N-terminally His-tagged GFPmut3b protein production could be induced.

Renilla luciferase vector
Primer pair RK005/RK006 were designed to PCR amplify the renilla luciferase gene along with the addition of restriction sites BamHI and XbaI from plasmid pRK5. The subsequent PCR product was designed so that it could be digested with BamHI and XbaI and ligated into pre-digested vector pWK-WT to form pRK6a vector in which Renilla Luciferase enzyme could be constitutively expressed.
The DNA of all inserts/constructs were verified by the sequencing service provided by Eurofins Genomics GmbH (Ebersberg, Germany). Primers AJW10 and AJW11 were used to sequence pSB1C3 based constructs and primers AJW77, AJW78, AJW322 and AJW376 were used to sequence either pHT01 or pHT01-ΔlacI based constructs. Primer WK3 was used to sequence the gfpmut3b constructs whilst primers RK001 and RK002 were used to sequence pPROEX HTb His-gfpmut3b.

Cell-free extract preparation
To prepare cell-free extracts, B. subtilis 168 cells were revived from glycerol stocks onto LB plates whilst B. subtilis WB800N cells were revived from glycerol stocks onto LB plates supplemented with kanamycin (Kan;10 µg/ml). Once streaked, plates were incubated for 48 h at 30°C. Individual colonies were inoculated into 5 ml 2× YTP medium and incubated for 10 h with shaking (180 rpm) at 30°C. The resultant cultures were diluted (1:500) into flasks containing 50 ml 2× YTP medium and incubated for 10 h with shaking (180 rpm) at 30°C. Resultant cultures were either harvested for cell lysis or, for larger scales of production, they were diluted (1:500) into flasks containing 500 ml 2× YTP medium and incubated for 10 h with shaking (180 rpm) at 30°C. To harvest cells, 500 ml cultures were centrifuged at 3,220g for 15 min. Cell pellets were re-suspended into 20 ml S30-A buffer (14 mM Magnesium (Mg) glutamate, 60 mM Potassium (K) glutamate, 50 mM Tris, 2 mM DTT, pH 7.7) and transferred into a pre-weighed 50 ml Falcon tube. Each 50 ml Falcon tube was centrifuged (2,000g, 10 min, 4°C), pellets washed with 20 ml S30-A buffer and subsequently re-centrifuged (2,000g, 10 min, 4°C) to form the final cell pellets in preparation for cell lysis. To determine the weight of the cell pellet, the weight of the 50 ml falcon tube was subtracted from the combined weight of the 50 ml tube and cell pellet. Pellets were stored at −80°C for no more than 48 h, prior to cell lysis.
To lyse the cells, pellets were defrosted on ice and re-suspended into 1 ml S30-A buffer per gram of cell pellet and aliquoted as 1 ml samples in 1.5 ml microtubes. Samples were sonicated on ice (3×40 s with 1-min cooling interval; output frequency: 20 kHz; amplitude: 50%) and then centrifuged (12,000g at 4°C for 10 min). The supernatants were removed, aliquoted at 500 μl into 2 ml screw cap tubes and incubated with shaking (180 rpm) at 37°C for either 0, 30 or 80 min. Post pre-incubation, samples were stored on ice and then centrifuged (12,000g at 4°C for 10 min). Supernatants were removed and were either aliquoted into 1.5 ml tubes that were stored on ice or into dialysis cassettes (GeBAflex-Maxi Dialysis Tubes −8 kDa MWCO, Generon) for dialysis into S30-B buffer (14 mM Mg-glutamate, 60 mM K-glutamate,~5 mM Tris, 1 mM DTT; pH 8.2) with stirring at 4°C for 3 h. Post-dialysis samples were centrifuged (12,000g at 4°C for 10 min), the extract supernatants from all conditions were aliquoted into 1.5 ml tubes, flash frozen in liquid nitrogen and stored at −80°C for use in cell-free reactions. The protein concentration of cell extracts was measured using a Bradford Assay (Biorad, CA, USA).

In vitro cell-free transcription-translation promoter library characterisation
Cell-free transcription-translation reactions were 10 μl in total and consisted of three parts mixed together in the indicated ratios: cell extract (33% v/v), optimised energy buffer (42% v/v) and DNA (25% v/ v). The final reaction conditions were: 8 mM Mg-glutamate, 160 mM K-glutamate, 1.5 mM each amino acid (except leucine −1.25 mM leucine), 50 mM HEPES, 1.5 mM ATP and GTP, 0.9 mM CTP and UTP, 0.2 mg/ml E. coli tRNA, 0.26 mM CoA, 0.33 mM NAD, 0.75 mM cAMP, 0.068 mM folinic acid, 1 mM spermidine, 2% PEG-8000, 30 mM 3-PGA and 10 nM plasmid DNA. 10 μl cell-free reactions were aliquoted into individual wells of 384-well plates (Griener bioone), measured using a Clariostar plate reader (BMG) with the following settings; Excitation 483 nm and Emission 530-30 nm. Plates were sealed, shaken prior to each reading cycle (500 rpm) and the plate reader was set to incubate the cell-free reactions at 30°C. The relative strength of the promoters was calculated from the rate of fluorescence increase during a phase of increasing GFPmut3b expression (20-80 min). The background fluorescence of cell-free reactions using the control plasmid (pHT01-ΔlacI), were subtracted and these data were normalised to the relative strength of prWK-WT which was denoted a relative strength of 1.

In vivo promoter library characterisation
Promoter library constructs selected for in vivo characterisation, along with the pHT01-ΔlacI empty vector control, were transformed into B. subtilis WB800N using the two-step transformation procedure as described previously (Cutting and Vander Horn, 1990) and transformants were selected on LB agar containing the appropriate antibiotics. This resulted in strains WB800N pHT01-ΔlacI (AJW25),

Plate reader characterisation
Promoter library strains were revived from glycerol stocks onto LB plates supplemented with the appropriate antibiotics and incubated for 48 h at 30°C. Individual colonies were inoculated into 5 ml 2x YTP medium with appropriate antibiotics and incubated overnight with shaking (180 rpm) at 30°C. The overnight cultures were diluted to an OD 600 nm of 0.05 in fresh 2x YTP with appropriate antibiotics and 100 μl aliquots loaded onto a 96-well black plate with clear flat bottoms (Greiner Bio-one, At; Cat #655076). Absorbance (600 nm) and fluorescence (Excitation 483 nm, Emission 530-30 nm) was measured every ten minutes at 30°C, with shaking at 700 rpm between each measurement in a BMG Clariostar plate reader (BMG, UK). Each strain was analysed using 3 independent cultures, with each culture being tested in triplicate. The relative strength of the promoters was calculated as the rate of fluorescence (GFPmut3b) per cell growth (OD 600 nm ) increase during a set time period (240-300 min). The background fluorescence of 2x YTP cell growth media and cells transformed with the negative control plasmid (pHT01-ΔlacI) were subtracted and these data were normalised to the relative strength of pWK-WT which was denoted an RPU of 1.

Flow cytometry characterisation
Promoter library strains were revived from glycerol stocks onto LB plates supplemented with the appropriate antibiotics and incubated for 48 h at 30°C. Three individual colonies were selected for each strain, separately inoculated into 5 ml 2x YTP medium with appropriate antibiotics and incubated overnight with shaking (180 rpm) at 30°C. Overnight cultures were diluted to an OD 600 nm of 1.0. 1 ml of diluted cell cultures were centrifuged (12,470g) and washed twice with 1 ml Phosphate Buffered Saline (1X PBS). Finally, cell pellets were resuspended into 1 ml PBS, then diluted (1:1000) into PBS before being loaded onto an Attune NxT (ThermoFisher Scientific, MA, USA) flow cytometer. The fluorescence (Geometric mean BL1-A; Ex. 488 nm, Em. 530/30) of at least 30,000 cells per sample were measured and these data were analysed using FlowJo (vX 10.1r5) software. The background fluorescence (BL1-A) of cells transformed with the negative control plasmid (pHT01-ΔlacI) were subtracted and these data were normalised to the relative strength of pWK-WT which was denoted a relative strength of 1.

Luciferase assay
Cell-free transcription-translation reactions were 10 μl in total and consisted of three parts mixed together in the indicated ratios: cell extract (33% v/v), optimised energy buffer (42% v/v) and DNA (25% v/ v). Cell-free reactions were setup to include either 10 nM (final concentration) of pHT01-ΔlacI or pHT01-ΔlacI-Renilla plasmid constructs. Cell-free reactions were incubated for 3 h at 30°C. Postincubation, cell-free reactions were transferred into white assay plates (Greiner Bio-One 96-well half-area) and assayed, according to manufacturer's guidelines, for detection of luciferase activity using a commercially available Renilla luciferase assay kit (Promega, WI, USA; Cat# E2810). Bioluminescence was measured using a Clariostar plate reader (BMG, UK) with the following settings: bioluminescence emission was measured at 480-80 nm and the plate reader was set to incubate the cell-free reactions at 30°C.

Cell-free workflow
The primary focus of this study was to develop a robust B. subtilis cell-free transcription-translation system that could be applied towards applications that are of interest to synthetic biologists and metabolic engineers such as cell-free protein synthesis, or regulatory element prototyping. To this end, our initial aim was to implement a standardised workflow of extract preparation and reaction optimisations that could be tailored towards the minimisation of cell extract batch variation and improvement of cell-free protein production yields. The workflow was developed for B. subtilis, though it incorporates aspects of several different established cell-free transcription-translation protocols (Shin and Noireaux, 2012;Shrestha et al., 2012;Sun et al., 2013) and consists of three phasesharvest cells, extract preparation and cell-free reaction optimisation (Fig. 1)..
During the cell growth phase B. subtilis strains were revived from glycerol stocks onto LB plates and incubated for 48 h at 30°C. From these, individually selected colonies were used to inoculate 5 ml cultures (2× YTP media) that were grown for 10 h with shaking at 30°C. These cultures were then diluted (1:500) into 50 ml 2× YTP media and grown for 10 h with shaking at 30°C, after which the cells were harvested during a late log phase of cell growth (Fig. S1a). Alternatively, if larger batches were required then cultures could be diluted once more (1:500) into 500 ml 2× YTP media and grown for a final 10 h with shaking at 30°C. For practical purposes, cells were harvested at the 50 ml growth stage since this produced enough cell extract for downstream characterisation experiments. These growth conditions were sufficient such that the final harvest OD 600 nm of both B. subtilis strains −168 (3.133 ± 0.033) and WB800N (2.722 ± 0.072) were consistent between batches of the same strain (Fig. S1b). Cell pellets were harvested through centrifugation, washed and stored at −80°C until the extract preparation phase.
During the extract preparation phase cell pellets were defrosted slowly on ice, re-suspended in 1 ml S30-A buffer per gram of cell pellet and each millilitre of cell and buffer mixture aliquoted into separate 1.5 ml microtubes. These samples were sonicated and then centrifuged to produce a clarified cellular extract. In order to produce cellular extracts that have optimal cell-free transcription-translation activity, clarified cellular extracts were aliquoted into distinct downstream processing groups. Cellular extracts were pre-incubated at 37°C with shaking (180 rpm) for either 0, 30 or 80 min. Similarly, to reports of E. R. Kelwick et al. Metabolic Engineering xx (xxxx) xxxx-xxxx coli cell-free extract preparation methods, the pre-incubation temperature influences the performance of cellular extracts in downstream cellfree transcription-translation reactions (Sushmita et al., 2015). We observed that B. subtilis WB800N extracts prepared with a preincubation temperature of 30°C displayed a reduction in cell-free transcription-translation activity in comparison to cell extracts prepared with a pre-incubation temperature of 37°C (Fig. S2). Therefore, we typically pre-incubated cell extracts at 37°C. Upon completion of the pre-incubation step the cell extracts were processed either with or without dialysis treatment at 4°C, with stirring, for 3 h. This resulted in the generation of six differently processed cellular extracts from each cell batch. The total protein content of these cellular extracts was analysed using Bradford assays. The total protein concentrations of B. subtilis 168 cellular extracts varied between cell batches and between the different extract processing methods, particularly for those extracts that were pre-incubated (Fig. S1c). In contrast, the total protein concentrations of B. subtilis WB800N cellular extracts were largely consistent across batches and processing methods (Fig. S1c). Processed cellular extracts were flash frozen in liquid nitrogen and stored at −80°C until used.
Processed extracts were assessed in terms of their cell-free transcription-translation reaction activities using a previously described (Sun et al., 2013) standard energy buffer (Table S3), and 10 nM of plasmid DNA. In this study, we chose to use the B. subtilis plasmid pHT01 as the backbone for our constructs in cell-free reactions since it has been previously validated as a stable expression vector for the production of recombinant proteins (Nguyen et al., 2007). The fluorescent reporter GFPmut3b was cloned into this plasmid to create the construct pHT01-gfpmut3b, such that the expression of gfpmut3b would be under the control of the lacI repressible P grac (σA) promoter. Addition of Isopropyl β-D-1-thiogalactopyranoside (IPTG) for inhibition of LacI repression enables the inducible expression of GFPmut3b ( Fig. 2a; Fig. 5b). However, when pHT01-gfpmut3b was tested in B. subtilis cell-free reactions, GFPmut3b production occurred regardless of IPTG induction (Fig. S3a). As such, the pHT01-gfpmut3b plasmid effectively resulted in the constitutive expression of gfpmut3b. Since the LacI repressor proteins are not present in the B. subtilis WB800N cell extract and are instead constitutively expressed from the lacI gene that is encoded into pHT01, it is likely that there are insufficient LacI repressor proteins during the early stages of the cellfree reaction. The unnecessary repressor lacI gene was removed, using PCR, from plasmids pHT01 and pHT01-gfpmut3b to create a negative control plasmid (pHT01-ΔlacI) and a constitutive gfpmut3b expression plasmid (pWK-WT) ( Fig. 2a; Fig. S3b; Fig. S4) that were subsequently used to test and compare cell-free reaction activities..
During the final phase of the workflow the most productive cellular Fig. 2. Characterisation of a Bacillus subtilis 168 cell-free transcription-translation system: (a) Schematic of the constructs used to characterise B. subtilis cell free systems. Circuits were visualised using Pigeon (Bhatia and Densmore, 2013). (b) Endpoints (5 h) of cell-free reactions using cell-extract batches prepared as indicated -including 0, 30 or 80 min preincubation at 37°C, followed either with or without dialysis treatment. The background fluorescence of cell-free reactions using the negative control plasmid (pHT01-ΔlacI), were subtracted. (c) Example time-course cell-free reactions using cell extracts that were prepared as indicated -including 0, 30 or 80 min pre-incubation at 37°C, followed either with or without dialysis treatment. Cell-free reactions contained either the negative control plasmid -pHT01-ΔlacI (indicated black) or pWK-WT (indicated green). Error bars denote standard error of the mean. extracts were improved through additional cell-free reaction optimisation steps. Emphasis was placed on changing the concentrations of magnesium glutamate and potassium glutamate in the energy buffer since these have previously been shown to have a significant influence on cell-free reaction activity (Cai et al., 2015;Sun et al., 2013). Upon completion of the workflow the extract preparation method and energy buffer composition that resulted in the greatest yield of GFPmut3b production could then be used for all subsequent batches. We initially used the workflow to characterise a B. subtilis 168 cell-free system.
3.2. Characterisation of a Bacillus subtilis 168 cell-free system B. subtilis 168 is an established and highly characterised strain whose genomic heritage spans several decades to the extent that its origins can be traced back to some of the earliest isolated legacy strains (Burkholder and Giles, 1947;Zeigler et al., 2008). B. subtilis 168 is a domesticated strain and as such, it is relatively easy to culture and genetically engineer (Guan et al., 2016;Zeigler et al., 2008). These characteristics have made B. subtilis 168 a suitable choice for a broad array of industrial biotechnology applications and more recently as a suitable host for synthetic biology . The universal utility of B. subtilis 168 suggests that the development of a B. subtilis 168 cell-free transcription-translation system would be a useful platform for synthetic biology and metabolic engineering applications.
In order to develop a B. subtilis 168 cell-free system, three independently generated batches of B. subtilis 168 were cultured, harvested and the resultant cell extracts processed using the cell-free workflow described in Fig. 1. These extracts were combined with the standard energy buffer and 10 nM (final concentration) of either the negative control plasmid (pHT01-ΔlacI) or the constitutive GFPmut3b expression plasmid (pWK-WT) to form cell-free reactions for testing. Replicate reactions were aliquoted into a 384 well plate and measured in parallel. Cell-free production of GFPmut3b was measured every ten minutes for five hours at 30°C using a Clariostar plate reader, with shaking before each measurement cycle to support oxygenation and mixing of the cell-free reactions. However, cell-free reactions using B. subtilis 168 cell extracts showed relatively little transcription and/or translation activity ( Fig. 2b; Fig. 2c). Indeed, end-point (5 h) analysis of GFPmut3b production across all cell-extract batches that were prepared as indicated -including 0, 30 or 80 min pre-incubation (37°C), followed either with, or without dialysis treatment, produced low and unreliable yields of GFPmut3b (Fig. 2b). GFPmut3b yields, calculated using a GFPmut3b calibration curve (Fig. S5c), ranged from effectively 0-0.011 µM ± 0.003 (pre-incubation for 0 min, without dialysis). A complete analysis of these data is shown in Table S4. Whilst alternative extract processing methods or energy buffers may improve cell-free activity, it is possible that despite the advantages of B. subtilis 168 in vivo, the strain is not intrinsically suitable for use in cell-free transcription-translation reactions. This could be due to the presence of endogenously expressed proteases and a resultant degradation of translated proteins. Indeed, previously reported B. subtilis cell-free systems were typically encumbered by a requirement to include protease inhibitors (Table 1). Rather than to optimise B. subtilis 168 cell-free activity through the addition of a cocktail of protease inhibitors, we decided to circumvent these limitations through the development of a B. subtilis cell-free system that uses cellular extracts from a protease deficient strain -B. subtilis WB800N (Nguyen et al., 2011).
3.3. The development of a B. subtilis WB800N cell-free system B. subtilis WB800N (MoBiTech, GmbH) is a commercially accessible strain that has been developed for the production and secretion of heterologous proteins (Nguyen et al., 2011). B. subtilis WB800N was engineered to be neomycin resistant and deficient for the expression of Table 1 Comparison of B. subtilis cell-free transcription-translation systems.

This study
Legault-Demare and Chambliss (1974) Leventhal and Chambliss (1979) Nes and Eklund (1983) Okamoto et al. (1985) Zaghloul and Doi (1987) Strain ( several proteases (nprE aprE epr bpr mpr::ble nprB::bsr Δvpr wprA::hyg cm::neo; NeoR) ( Fig. 3a). Similarly, to the generation of B. subtilis 168 extracts, three independently generated batches of B. subtilis WB800N cells were cultured, harvested and the resultant extracts were processed using the cell-free workflow described in Fig. 1. Cell-free reactions using B. subtilis WB800N cell-extracts were most active during the first 0-150 min and were generally more active, in terms of GFPmut3b production, than B. subtilis 168 cell-extracts Fig. 3. Development of a Bacillus subtilis WB800N cell-free transcription-translation system. (a) B. subtilis WB800N is an engineered strain in which the indicated proteases (X) have been knocked-out. Adapted from (Westers et al., 2004). (b) Endpoints (5 h) of cell-free reactions using cell extract batches prepared as indicated -including 0, 30 or 80 min preincubation at 37°C, followed either with or without dialysis treatment. The background fluorescence of cell-free reactions using the negative control plasmid (pHT01-ΔlacI), were subtracted. (c) Representative time-courses of cell-free reactions using cell-extracts that were prepared as indicated -including 0, 30 or 80 min pre-incubation at 37°C, followed either with or without dialysis treatment. Cell-free reactions contained either the negative control plasmid -pHT01-ΔlacI (indicated black) or pWK-WT (indicated green). (d) Optimisation of cell-free buffer components: magnesium glutamate and potassium glutamate. These data are representative of endpoint (5 h) analysis of cell-free reactions from three independently prepared extracts. The background fluorescence of cell-free reactions using the negative control plasmid (pHT01-ΔlacI), were subtracted. (e) Endpoints (5 h) of optimised cell-free reactions that include a range of different plasmid DNA (pWK-WT) concentrations. The background fluorescence of cell-free reactions using the negative control plasmid (pHT01-ΔlacI), were subtracted. Error bars denote standard error of the mean.
( Fig. 3b; Fig. 3c). Based upon an analysis of end-point (5 h) GFPmut3b cell-free reaction yields, WB800N cell-extracts (Fig. 3b) produced higher and more consistent yields of GFPmut3b than B. subtilis 168 cell-extracts (Fig. 2b). WB800N cell-free GFPmut3b production yields, calculated using a GFPmut3b calibration curve (Fig. S5c), ranged from 0.041 µM ± 0.008 (pre-incubation for 80 min, without dialysis) to 0.116 µM ± 0.009 (pre-incubation at 37°C for 80 min, followed by dialysis) (Table S4).. In continuation of the cell-free workflow (Fig. 1) additional experiments were undertaken to further improve B. subtilis WB800N cellfree transcription-translation reactions through changes in the composition of the energy buffer. The concentrations of magnesium glutamate (Mg-glutamate) and potassium glutamate (K-glutamate) in the standard energy buffer were altered since these have been previously shown to have a significant influence on cell-free reaction activity. Previous reports have demonstrated that glutamate is utilised in the TCA cycle of cellular extracts and is therefore able to serve as an energy substrate that supports ATP generation and protein production (Jewett et al., 2008). The processing conditions that included 80 min pre-incubation at 37°C and dialysis produced the most active cellular extracts and therefore, these conditions were used for the generation of three additional B. subtilis WB800N extract batches. These processed cell extracts were combined with 10 nM (final concentration) of either pHT01-ΔlacI (control) or pWK-WT plasmid DNA and energy buffers that were similar in composition to the standard energy buffer, except that they contained different combinations of K-glutamate (40−160 mM) and Mg-glutamate (4 mM−12 mM) concentrations. In comparison to the standard energy buffer, which enabled the cell-free production of 0.281 µM ± 0.019 GFPmut3b, an optimised energy buffer that included 160 mM K-glutamate and 8 mM Mg-glutamate resulted in an improved GFPmut3b production yield of 0.500 µM ± 0.052 ( Fig. 3d; Table S3; Table S5).
An additional batch of B. subtilis WB800N cell extract was processed using our improved protocol. This new batch of cell extract was used to setup several cell-free reactions that when tested with a range of different pWK-WT plasmid DNA concentrations produced up to 0.848 µM ± 0.047 of GFPmut3b (Fig. 3e). In these experiments, there was generally a linear relationship between DNA concentration (pWK-WT) and cell-free GFPmut3b production however, when the final DNA concentration was increased above 8 nM this linearity did not continue. Essentially, further increases in DNA concentration above 8 nM did not result in significant increases in GFPmut3b production. It is possible that relatively high concentrations of plasmid DNA pose a maximal usage of available transcription (e.g. RNA polymerases) and/or translation (e.g. ribosomes) machinery in the cell extract that is limiting any further improvements in cell-free reaction performance (Ceroni et al., 2015). As a preliminary test, we explored whether an increase in the proportion of cellular extract from 33% (v/ v) to 50% (v/v) of the total cell-free reaction volume would improve cell-free transcription-translation activity. As expected, increasing the volume of cellular extract (to 50% v/v) and as a likely consequence, increasing the availability of transcription and/or translation machinery, resulted in a greater level of cell-free reaction activity (Fig. S6). In separate experiments, we observed that the addition of E. coli tRNAs improved the activity of B. subtilis WB800N cell-free transcriptiontranslation reactions but were not essential (Fig. S7). Thus, whilst we cannot discount the possibility that further improvements in B. subtilis WB800N cell-free activity might be hampered by the availability of core cellular machinery (e.g. active ribosomes) in the cell-free extract, these data suggest that at least some of the translational resources (e.g. tRNAs) may not be a significant limiting factor.
Future studies may also need to consider additional factors that might be impacting B. subtilis WB800N cell-free reaction activity. For instance, there are reports that changes in pH as a result of the buildup of glycolytic lactate and acetate production, as well as the production of inorganic phosphates during cell-free reactions can also be deleterious to cell-free activity Noireaux, 2015b, 2014). These limiting factors may be mitigated through changes in the energy source that is used. For example, Maltose is a novel energy source that enables the recycling of inhibitory inorganic phosphates in E. coli cellfree systems (Caschera and Noireaux, 2014). Therefore, in future studies, it may be possible to further improve B. subtilis cell-free activity through an investigation of these potentially inhibitory factors and to assess whether changes in the energy buffer composition might mitigate any limiting effects.

Generation of an engineered promoter library
In order to improve the ability of synthetic biologists and metabolic engineers to precisely fine tune gene expression there is an increasing interest towards expanding the availability of characterised genetic regulatory elements (e.g. promoters) (Guiziou et al., 2016;Moore et al., 2016;Patron et al., 2015). As demonstration of the applicability of B. subtilis cell-free transcription-translation systems for regulatory element prototyping we applied our improved B. subtilis WB800N cellfree system towards the characterisation of an engineered B. subtilis promoter library that enables a range of transcriptional activities in vitro (cell-free) and in vivo. The engineered promoter library was created using degenerate oligonucleotides that were designed to bind to the −35 and −10 boxes of the P grac (σA) promoter in plasmid pWK-WT, and introduce, through an inverted PCR, changes into the promoter sequence (Fig. 4a). This strategy should result in enough random changes to both boxes (−10 and −35) to give a theoretical library size of up to 4 12 (1,6777,216) different engineered promoters. Several library clones were screened via sequencing and those with sequence changes that differed from the reference wildtype P grac promoter, which we renamed as prWK-WT, were retained for further studies. The promoter library sequences obtained are shown in Fig. S8. The plasmid -pWK-Δbox was also engineered as an additional negative control in which primers were designed to remove, via PCR from plasmid pWK-WT, both the −35 and −10 boxes of the P grac (σA) promoter, as well as the sequence between them. Thus, plasmid pWK-Δbox did not promote gfpmut3b expression ( Fig. 4b; Fig. S9; Fig. S10b; Fig. S11; Table S7; Table S8)..
An initial screen of cell-free GFPmut3b production from 58 different engineered promoter constructs was carried out using the improved B. subtilis WB800N cell-free system. Our initial screening identified several promoters that displayed a range of different transcriptional strengths. Engineered promoters that resulted in the highest levels of gfpmut3b expression were selected for further analysis (Fig. S9). Of these, clones: pWK1, pWK28, pWK76, pWK104, pWK105, pWK118, pWK120, pWK301, pWK319, pWK603, and pWK609, were characterised for both in vitro (cell-free transcription-translation) and in vivo gfpmut3b expression.
Similarly to previous reports (Chappell et al., 2013;Kelly et al., 2009) we assessed the relative strength of these engineered promoters using cell-free transcription-translation (in vitro), plate reader (in vivo) and flow cytometry (in vivo) assays (Fig. 4b). These assays are described in more detail in the materials and methods section. Briefly, for cell-free transcription-translation characterisation of the promoter library the relative strength of the promoters was calculated from the rate of fluorescence increase during a phase of increasing GFPmut3b expression (20-80 min) and then these rate change data were normalised (including removal of the background signal) to the relative strength of prWK-WT. Whilst, for in vivo analysis, promoter library strains (B. subtilis WB800N) were cultured in 96-well plates and assayed using a Clariostar plate reader (Fig. S10). The relative strength of the promoters was calculated as the rate of fluorescence (GFPmut3b) increase per cell growth (OD 600 nm ) during a set time period (240-300 min). and then these rate change data were normalised (including removal of the background signal) to the relative strength of prWK-WT. Finally, as an additional assessment, the promoter library strains (B. subtilis WB800N) were cultured overnight, washed in PBS and loaded into an Attune NxT flow cytometer for analysis of GFPmut3b fluorescence (Geometric mean BL1-A) (Table S8.). These data were subsequently normalised (including removal of the background signal) to the relative strength of prWK-WT.
We observed significant differences in activity between promoters that were largely similar in sequence. For example, the sequences of prWK104 and prWK1 differ by only two base changes (−35 box: T > C; −10 box: G > A) yet, on relative terms, prWK104 is roughly four times stronger than prWK1. It is likely, that these base changes are influencing the biophysical interaction (e.g. binding affinity) between the promoter region on the plasmid DNA and the transcriptional machinery (e.g. RNA polymerase and sigma factor) (Guiziou et al., 2016). Interestingly, the general pattern of relative promoter strengths in the engineered library were similar both in vitro (cell-free) and in vivo ( Fig. 4b; Table S7). Comparability between the relative activity of genetic regulatory elements tested in cell-free transcription-translation reactions and in vivo has been previously demonstrated in E. coli (Chappell et al., 2013;Sun et al., 2014). Comparability between cellfree and in vivo characterised DNA regulatory elements is important within a systematic design contextthrough which, it is envisioned that cell-free workflows may rapidly provide the characterisation data required to rationally select a smaller number of designs for final testing in vivo. Therefore, whilst further investigations are needed to assess the in vivo comparability of in vitro characterised B. subtilis genetic regulatory elements, characterisation of our engineered promoter library suggests that B. subtilis cell-free systems are a viable platform for regulatory element prototyping.
3.5. Characterisation of a B. subtilis WB800N-pHT01 cell extract for inducible gene expression Genetic circuit engineering lies at the foundations of synthetic biology (Collins et al., 2000;Elowitz and Leibler, 2000) and there is a growing interest in utilising cell-free transcription-translation systems to iterate increasingly more complex genetic circuit designs (Niederholtmeyer et al., 2015;Noireaux et al., 2003;Zhang et al., 2016). Therefore, it is likely that as the availability of characterised B. subtilis regulatory elements increases there will be an increase in the potential for using B. subtilis cell-free transcription-translation systems to support the development of B. subtilis-based genetic circuits (Jeong et al., 2015). Consequently, as an initial step towards the emergence of genetic circuit prototyping in B. subtilis cell-free systems we characterised an inducible expression system. The protease deficient strain -B. subtilis WB800N was previously transformed with plasmid pHT01 to create the engineered strain -WB800N-pHT01 (AJW5) which constitutively expresses the lacI gene ( Fig. 5a) (Webb et al., 2016). Three independent batches of B. subtilis WB800N-pHT01 cell extracts were generated using the same method that improved the cell-free activity of B. subtilis WB800N cell extracts (pre-incubation at 37°C for 80 min, followed by dialysis). As expected, the presence of LacI repressor protein within B. subtilis WB800N-pHT01 cell extracts was sufficient to inhibit (turn "OFF") GFPmut3b expression from pHT01based plasmids in cell-free transcription-translation reactions ( Fig. 5b;  Fig. 5c; Fig. S12). Likewise, addition of 0.0625-1 mM IPTG for inhibition of LacI repression resulted in a robust induction (turn "ON") of GFPmut3b expression from pHT01-gfpmut3b plasmidproducing a fluorescent signal that was 117-147 fold above background ( Fig. 5b; Fig. 5c). Whilst, there was a modest induction increase between the lowest (0.0625 mM) and highest (0.5-1 mM) concentrations of IPTG that were tested, it may be possible to achieve a larger dynamic range of induction levels if a broader range of IPTG concentrations are used. Nevertheless, these inducible expression data suggest that it may be possible, in future studies, to use these B. subtilis cell-free systems to characterise simple genetic circuits..

Characterisation of Renilla luciferase activity in B. subtilis WB800N cell-free reactions
The luciferase gene from Renilla reniformis (sea pansy) encodes a decarboxylating enzyme that is capable of catalyzing the oxidation of Coelenterazine to Coelenteramide. As a consequence, cabon dioxide (CO 2 ) and bioluminescence (480 nm) are produced (Lorenz et al., 1991;Matthews et al., 1977). The bioluminescence capabilities of Renilla luciferase have been extensively characterised and repurposed for use in an array of contexts and biological assays (Hampf and Gossen, 2006). We carried out cell-free protein expression and characterisation of Renilla luciferase activity in B. subtilis WB800N cell-free extracts. Cell-free reactions including either 10 nM (final concentration) of pHT01-ΔlacI or pHT01-ΔlacI-renilla plasmid DNA were incubated at 30°C for three hours prior to the addition of the luciferase assay buffer and substrate. Bioluminescence (480-80 nm) was detected in cell-free reactions that expressed Renilla luciferase from plasmid pHT01-ΔlacI-renilla and as expected control reactions (with plasmid pHT01-ΔlacI) produced negligible bioluminescence (Fig. 6b, Fig. 6c). These experiments were carried out in order to demonstrate the potential applicability of B. subtilis cell-free transcription-translation reactions for cell-free protein production and in situ Schematic displaying the sequences of engineered promoters that were derived from PCR reactions involving degenerate primers that target the −10 and −35 boxes within the wildtype P grac (σA) promoter (prWK-WT) (b) For in vitro cell-free characterisation, the relative strength of the promoters was calculated from the rate of fluorescence increase during a phase of increasing GFPmut3b expression (20-80 min). The background fluorescence of cell-free reactions using the negative control plasmid (pHT01-ΔlacI), were subtracted and these data were normalised to the relative strength of prWK-WT which was denoted a relative strength of 1. For in vivo plate reader characterisation, the relative strength of the promoters was calculated as the rate of fluorescence (GFPmut3b) per cell growth (OD 600 nm ) increase during a set time period (240-300 min). The background fluorescence of 2x YTP cell growth media and cells transformed with the negative control plasmid (pHT01-ΔlacI) were subtracted and these data were normalised to the relative strength of pWK-WT which was denoted a relative strength of 1. For in vivo flow cytometry characterisation, the fluorescence (Geometric mean BL1-A; Ex. 488 nm, Em. 530/30) was measured using an Attune NxT flow cytometer and were analysed using FlowJo (vX 10.1r5) software. The background fluorescence of cells transformed with the negative control plasmid (pHT01-ΔlacI) were subtracted and these data were normalised to the relative strength of pWK-WT which was denoted a relative strength of 1. Error bars denote standard error of the mean. characterisation of enzyme performance. tableIt is likely that as cellfree protein synthesis driven metabolic engineering approaches develop the parallel characterisation of enzymes, that are encoded within DNA libraries, may enable the rapid optimisation of biosynthetic pathways (Dudley et al., 2015;Karim and Jewett, 2016)..

Conclusions
B. subtilis is an established model organism of broad importance to microbiology, synthetic biology and industrial biotechnology. Therefore, the development of B. subtilis cell-free transcription-translation systems are desirable since, much like existing cell-free transcription-translation systems, they could be applied to several applications. Yet, despite their potential B. subtilis cell-free transcriptiontranslation systems have been largely neglected. Previously reported B. subtilis cell-free transcription-translation systems were typically encumbered by a range of different factors that significantly hindered the accessibility of the methods used to generate them and their capabilities (e.g. poor reaction dynamics and low cell-free protein production yields) (Table 1).
Herein, we report on the development of several B. subtilis cell-free systems that unlike previously reported methods do not require the preparation or use of exogenous mRNAs, ribosomes, DNAse or protease inhibitors. Consequently, we report that our method is much more accessible and easier to carry out in a shorter time frametypically several batches of cell extract can be generated in just a few days. Additionally, in contrast to previous reports, we describe the use and improvement of a relatively more efficient energy regeneration system, based on 3-phosphoglycerate (3-PGA) and optimised concentrations of magnesium and potassium glutamate, that is now typically used in E. coli cell-free transcription-translation reactions. In combination these improvements have enabled the development of an improved B. subtilis WB800N system that is capable of robust cellfree transcription-translation reactions that last for several hours and can produce up to 0.8 µM GFPmut3b, which is a~72× fold-improvement when compared with a B. subtilis 168 cell-free transcriptiontranslation system (0.011 µM GFPmut3b). However, additional improvements are needed to increase B. subtilis cell-free activity to comparable levels of recently published E. coli cell-free transcriptiontranslation systems (production of over 40 µM of reporter protein) Noireaux, 2015b, 2014). Conversely, an examination of developments in E. coli or other cell-free transcription systems may provide insights into how further improvements in B. subtilis cell-free activity may be achieved. In particular, an examination of alternative energy sources (e.g. maltose, maltodextrin or hexametaphosphate), cofactor regeneration systems and an analysis of the availability of the cellular machinery (e.g. ribosomes) in cell extract batches may be the most significant priorities for future studies.
Whilst, further improvements in B. subtilis cell-free activity are desirable we demonstrate the applicability of our reported B. subtilis WB800N cell-free transcription-translation system towards regulatory element prototyping. Essentially, we engineered several promoters, derived from the wild-type P grac (σA) promoter, that display a range of comparable in vitro and in vivo transcriptional activities. Thus, we anticipate that these cell-free systems will be useful to applications  (a) Schematic depicts the cell-free expression and enzymatic activity of Renilla luciferase within B. subtilis WB800N cell-free transcription-translation reactions. Cell-free reactions including either 10 nM (final concentration) of pHT01-ΔlacI or pHT01-ΔlacIrenilla were incubated at 30°C for three hours prior to the addition of the luciferase assay buffer and substrate. (b) Time course analysis of Renilla luciferase activity within Cell-fee reactions. Luminesence (480-80 nm) was measured, post-2-second delay, for 10 s using a Clariostar plate reader. (c) Integrated analysis was calculated as the summation of luminescence, throughout the 10 s measurement cycle, for each sample. Error bars denote standard error of the mean.
such as the engineering of in vivo metabolic pathways or genetic circuits that require characterised regulatory elements (e.g. promoters) in order to rationally fine tune gene expression. Additionally, as a step towards future applications for B. subtilis cell-free systems, we described the characterisation of an inducible expression system (a precursor to genetic circuit prototyping) and we also characterised the activity of Renilla luciferase (a model enzyme). More broadly, we envision that these improved B. subtilis cell-free transcription-translation systems will spur a renewal in efforts to continue the development of these systems for the benefit of an array of synthetic biology and metabolic engineering applications.