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

Richard Kelwick1,2#, Alexander J. Webb1,2#, James T. MacDonald1,2, and Paul S. Freemont1,2*

1Centre for Synthetic Biology and Innovation, Imperial College London, London SW7 2AZ, UK, 2Section of Structural Biology, Department of Medicine, Imperial College London, London SW7 2AZ, UK

#Joint First Authors *To whom correspondence should be addressed:

Paul Freemont Section of Structural BiologyDepartment of MedicineSir Alexander Fleming BuildingSouth Kensington Campus Exhibition Road London  SW7 2AZ  UK Email: p.freemont@imperial.ac.uk Tel: +44 (0) 207 594 5327 ABSTRACT

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 Pgrac (σA) 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.

Keywords: Synthetic biology; Bacillus subtilis; cell-free transcription-translation; regulatory element prototyping; promoter library; luciferase assay

Running Title: A Bacillus subtilis cell-free system for synthetic biology

Abbreviations: ATP, adenosine triphosphate; CTP, cytidine triphosphate; GTP, guanosine triphosphate; UTP, uridine triphosphate; GFPmut3b, green fluorescent protein mut3b variant; Mg, magnesium; K, potassium; tRNA, transfer ribonucleic acid; 3-PGA, 3-phosphoglycerate; NAD, nicotinamide; CoA, coenzyme A; cAMP, adenosine 3’,5’-cyclic monophosphate; DTT, dithiothreitol.

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1. 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 transcription–

translation 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

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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 (Caschera and

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

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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).

2. Materials and methods

2.1. Bacterial strains, plasmids and growth conditions

Bacterial strains used in this study are listed in Table S1. E. coli strains were grown

in Luria-Bertani (LB) medium at 37oC whilst B. subtilis strains were grown in 2x YTP

medium (31 g/L 2x YT, 40 mM potassium phosphate dibasic, 22 mM potassium

phosphate monobasic) at 30oC. When applicable, the medium was supplemented

with the following antibiotics: E. coli cultures - ampicillin (Amp) 100 μg/ml;

chloramphenicol (Cam) 50 μg/ml; kanamycin (Kan) 35 μg/ml; B. subtilis WB800N

cultures – chloramphenicol 5 μg/ml; kanamycin 10 μg/ml. Kanamycin is used to

select for the neomycin (Neo) resistance gene in B. subtilis WB800N.

2.2. 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

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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 NEB10-beta and the colonies

cultured on plates incubated at either 30oC or 37oC. 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, self-ligated 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

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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 pRK2 – a 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

pRK6 – a 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-Δ lacI based

constructs. Primer WK3 was used to sequence the gfpmut3b constructs whilst

primers RK001 and RK002 were used to sequence pPROEX HTb His-gfpmut3b.

2.3. 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 30oC. Individual colonies were inoculated into 5 ml 2x YTP

medium and incubated for 10 hr with shaking (180 rpm) at 30°C. The resultant

cultures were diluted (1:500) into flasks containing 50 ml 2x YTP medium and

incubated for 10 h with shaking (180 rpm) at 30°C. Resultant cultures were either

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harvested for cell lysis or, for larger scales of production, they were diluted (1:500)

into flasks containing 500 ml 2x YTP medium and incubated for 10 hr with shaking

(180 rpm) at 30°C. To harvest cells, 500 ml cultures were centrifuged at 3,220 g for

15 minutes. Cell pellets were re-suspended into 20 ml S30-A buffer (14 mM

Magnesium (Mg) glutamate, 60 mM Potassium (K) glutamate, 50 mM Tris, 2mM

DTT, pH 7.7) and transferred into a pre-weighed 50 ml Falcon tube. Each 50 ml

Falcon tube was centrifuged (2,000 g, 10 min, 4oC), pellets washed with 20 ml S30-A

buffer and subsequently re-centrifuged (2,000 g, 10 min, 4oC) 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 -80oC 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 x 40 seconds with 1-minute cooling interval;

output frequency: 20 KHz; amplitude: 50%) and then centrifuged (12,000 g 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 (12,000 g 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, 1mM DTT; pH 8.2) with stirring at 4°C for 3 hr. Post-

dialysis samples were centrifuged (12,000 g at 4 °C for 10 min), the extract

supernatants from all conditions were aliquoted into 1.5 ml tubes, flash frozen in

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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).

2.4. Cell-free transcription-translation reactions

Cell-free reactions were 10 μl in total and consisted of three parts mixed together in

the indicated ratios: cell extract (33% or 50% v/v), energy buffer (42% v/v) and DNA

(25% or 8% v/v). The final reaction conditions were: 4-12 mM Mg-glutamate, 40-160

mM K-glutamate, 1.5 mM each amino acid (except leucine - 1.25 mM leucine) [RTS

Amino Acid Sampler, 5-Prime, DE], 50 mM HEPES, 1.5 mM ATP and GTP, 0.9 mM

CTP and UTP, 0-0.2 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% (w/v) PEG-8000, 30 mM 3-PGA

and 0-12 nM plasmid DNA. 10 μl cell-free reactions were aliquoted into individual

wells of 384-well plates (Griener bio-one, NC, USA) and measured using a Clariostar

plate reader (BMG, UK) 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 30oC.

2.5. 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-

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free reactions were aliquoted into individual wells of 384-well plates (Griener bio-

one), 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 30oC. The relative strength of the promoters was calculated from the

rate of fluorescence increase during a phase of increasing GFPmut3b expression

(20 min – 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.

2.6 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), WB800N pWK-

WT (WK1), WB800N pWK-Δbox (WK2), WB800N pWK1 (WK3), WB800N pWK28

(WK4), WB800N pWK76 (WK5), WB800N pWK104 (WK6), WB800N pWK105

(WK7), WB800N pWK118 (WK8), WB800N pWK120 (WK9), WB800N pWK301

(WK10), WB800N pWK319 (WK11), WB800N pWK603 (WK12) and WB800N

pWK609 (WK13).

2.6.1 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 30ºC.

Individual colonies were inoculated into 5 ml 2x YTP medium with appropriate

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antibiotics and incubated overnight with shaking (180 rpm) at 30ºC. The overnight

cultures were diluted to an OD600nm 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 30ºC,

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 (OD600nm) increase

during a set time period (240 min – 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.

2.6.2 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 30ºC. 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 30ºC. Overnight cultures were diluted to an OD600nm of 1.0. 1 ml of diluted cell

cultures were centrifuged (12,470 g) and washed twice with 1ml Phosphate Buffered

Saline (1X PBS). Finally, cell pellets were re-suspended into 1ml 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. 488nm,

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)

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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.

2.7. GFPmut3b expression and purification

A culture of glycerol stocked E. coli containing plasmid pROEX HTb His-gfpmut3b

(pRK2) was used to inoculate 5 ml LB supplemented with 100 µg/ml ampicillin. The

culture was grown with shaking (180 rpm) for 16 h at 37oC. Subsequently, the culture

was diluted (1:500) into 500 ml LB supplemented with 100 µg/ml ampicillin, and 1

mM IPTG and grown with shaking (180 rpm) for 24 h at 37oC. The cell pellet was

harvested through centrifugation at 3,220 g for 15 minutes and stored at -80oC.

Pellets were defrosted on ice and re-suspended into 1 ml re-suspension buffer (50

mM Na2HPO4, 100 mM NaCl titrated to pH 8 with HCl/NaOH) per gram of cells. Re-

suspended cells were sonicated (3 x 40 seconds with 1-minute cooling interval;

output frequency: 20 KHz, Amplitude: 50%) and centrifuged (2000 g, 10 min, 4oC) to

produce a clarified extract. His–GFPmut3b was purified from the clarified extract

using a Ni-NTA column with wash buffer (50 mM Na2HPO4, 100 mM NaCl, 25 mM

Imidazole, titrated to pH 8 with HCl/NaOH) and elution buffer (50 mM Na2HPO4, 100

mM NaCl, 500 mM Imidazole, titrated to pH 8 with HCl/NaOH). The eluted His–

GFPmut3b purified fraction was dialysed using dialysis tubing (MWCO 12-14,000

kDa) suspended in 1 L dialysis buffer (20 mM HEPES, 100 mM NaCl, dH20 pH 8 with

KOH) overnight with stirring, in the dark, at 4oC. The protein concentration of the

purified His–GFPmut3b was determined using a Nanodrop ND-1000

spectrophotometer (Thermo Scientific). GFP purification samples were analysed via

sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE) using 4-

12% Bis-Tris gels (NuPAGE Novex, Lifetech), followed by either coomassie blue

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staining or western blot analysis with HRP-conjugated GFP-specific polyclonal

antibody (1:4,000 dilution; #A10260, Thermo Fisher Scientific Ltd, UK). Western

blots were developed by enhanced chemiluminence (ECL).

2.8. GFPmut3b calibration curve

GFPmut3b calibration curve reactions (10 μl) were setup similarly to typical cell-free

reactions, but without DNA, and consisted of three parts mixed together in the

indicated ratios: cell extract (33% v/v), buffer (42% v/v) and purified His-GFPmut3b

(25% v/v). The GFPmut3b calibration solutions contained: 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, 0-0.2 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 0-1.09 µM His–GFPmut3b. The

calibration curve reactions were aliquoted into 384-well plates (Greiner Bio-One, At)

and measured using a Clariostar plate reader (BMG, UK) with the following settings:

Excitation 483 nm, Emission 530-30 nm.

2.9. 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 hours at 30oC. Post-incubation,

cell-free reactions were transferred into white assay plates (Greiner Bio-One 96-well

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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 30oC.

3. Results and discussion

3.1. 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 phases – harvest 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 30oC. From these, individually selected

colonies were used to inoculate 5 ml cultures (2x YTP media) that were grown for 10

hours with shaking at 30oC. These cultures were then diluted (1:500) into 50 ml 2x

YTP media and grown for 10 hours with shaking at 30oC, after which the cells were

harvested during a late log phase of cell growth (Fig. S1a). Alternatively, if larger

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batches were required then cultures could be diluted once more (1:500) into 500 ml

2x YTP media and grown for a final 10 hours with shaking at 30oC. 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 OD600nm 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 -80oC 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 37oC with shaking (180 rpm) for either 0, 30

or 80 minutes. Similarly, to reports of E. coli cell-free extract preparation methods,

the pre-incubation temperature influences the performance of cellular extracts in

downstream cell-free transcription-translation reactions (Sushmita et al., 2015). We

observed that B. subtilis WB800N extracts prepared with a pre-incubation

temperature of 30oC displayed a reduction in cell-free transcription-translation activity

in comparison to cell extracts prepared with a pre-incubation temperature of 37oC

(Fig. S2). Therefore, we typically pre-incubated cell extracts at 37oC.

Upon completion of the pre-incubation step the cell extracts were processed either

with or without dialysis treatment at 4oC, with stirring, for 3 hours. This resulted in the

generation of six differently processed cellular extracts from each cell batch. The

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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 -80oC 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 Pgrac (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 cell-free reaction. The unnecessary repressor lacI

gene was removed, using PCR, from plasmids pHT01 and pHT01-gfpmut3b to

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create a negative control plasmid (pHT01-∆lacI) 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 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.

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Fig. 1. Cell-free workflow. A graphical depiction of the workflow used to develop

and characterise B. subtilis cell-free systems.

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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 (Harwood et al., 2013). 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-∆lacI) 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 30oC 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 minutes pre-

18

incubation (37oC), 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 µM to 0.011 µM

±0.003 (pre-incubation for 0 minutes, 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).

19

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 minutes pre-incubation at 37oC, 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 minutes pre-incubation at 37oC, followed either with or without

dialysis treatment. Cell-free reactions contained either the negative control plasmid –

20

pHT01-ΔlacI (indicated black) or pWK-WT (indicated green). Error bars denote

standard error of the mean.

21

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 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 minutes and were generally

more active, in terms of GFPmut3b production, than B. subtilis 168 cell-extracts (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 minutes, without dialysis) to 0.116 µM

±0.009 (pre-incubation at 37oC for 80 minutes, followed by dialysis) (Table S4).

In continuation of the cell-free workflow (Fig. 1) additional experiments were

undertaken to further improve B. subtilis WB800N cell-free 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

22

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 minutes pre-incubation at 37oC 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-∆lacI

(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

23

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

transcription-translation 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 build-up 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 (Caschera and 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 cell-free 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

24

assess whether changes in the energy buffer composition might mitigate any limiting

effects.

25

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.,

26

2004). (b) Endpoints (5 h) of cell-free reactions using cell extract batches prepared

as indicated - including 0, 30 or 80 minutes pre-incubation at 37oC, 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 minutes pre-incubation at 37oC,

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.

27

3.4. 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 cell-free 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 Pgrac 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 Pgrac (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

28

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 min –

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 (OD600nm) during a set time period (240 min – 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.

29

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 cell-free and in vivo characterised

DNA regulatory elements is important within a systematic design context – through

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.

30

Fig. 4. Characterisation of an engineered Bacillus subtilis promoter library. (a)

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 Pgrac (σA) promoter (prWK-WT) (b) For in vitro cell-free characterisation,

the relative strength of the promoters was calculated from the rate of fluorescence

31

increase during a phase of increasing GFPmut3b expression (20 min – 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 (OD600nm) increase during a set time

period (240 min – 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. 488nm, 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.

32

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 37oC for 80

minutes, 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 pHT01-based 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 plasmid – producing 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)

33

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.

34

35

Fig. 5. Characterisation of a Bacillus subtilis WB800N-pHT01 cell-free

transcription-translation system for inducible gene expression. (a) Schematic

depicts the generation of B. subtilis WB800N-pHT01 cell extracts for inducible gene

expression (b) Plasmid pHT01-gfpmut3b enables IPTG inducible gfpmut3b

expression within B. subtilis WB800N-pHT01-based cell-free transcription-translation

reactions. (c) Endpoint (5 h) characterisation of IPTG induced gene expression from

plasmid pHT01-gfpmut3b (10 nM final concentration) in B. subtilis WB800N-pHT01

cell-free transcription-translation reactions. The background fluorescence of cell-free

reactions using the negative control plasmid (pHT01), and the indicated range of

IPTG were subtracted. Error bars denote standard error of the mean. Student t-test,

*P<0.05.

36

3.6. 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 (CO2) 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-∆lacI or pHT01-∆lacI-renilla

plasmid DNA were incubated at 30oC 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-∆lacI-renilla

and as expected control reactions (with plasmid pHT01-∆lacI) 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 characterisation of enzyme

performance. It is likely that as cell-free protein synthesis driven metabolic

engineering approaches develop the parallel characterization of enzymes, that are

encoded within DNA libraries, may enable the rapid optimisation of biosynthetic

pathways (Dudley et al., 2015; Karim and Jewett, 2016).

37

Fig. 6. Characterisation of Renilla luciferase in B. subtilis WB800N cell-free

reactions. (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-

∆lacI or pHT01-∆lacI-renilla were incubated at 30oC 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 seconds using a Clariostar plate reader. (c)

Integrated analysis was calculated as the summation of luminescence, throughout

the 10 second measurement cycle, for each sample. Error bars denote standard

error of the mean.

38

4. 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 transcription-translation

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).

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(s) WB800N 168T+ - ATCC 6633 1A292 168Reaction type

Coupled transcription-

translation

mRNA-directed

Coupled transcription-

translation

mRNA-directed Coupled transcription-

translation

Coupled transcription-

translationEnergy source(s)

3-PGA, glutamate

Phosphoenolpyruvate, pyruvate kinase

- Phosphoenolpyruvate, pyruvate

kinase

Phosphoenolpyruvate, pyruvate kinase

Phosphoenolpyruvate, pyruvate kinase

Treatment Not required Protease inhibitors

DNAse treatment of ribosomes

2-mercaptoethanol

Exogenous ribosomes,

protease inhibitors

Exogenous ribosomes,

protease inhibitors

Reaction yield

~0.8 µM GFPmut3b

Incorporation of 826 pmoles [14C ]leucine

or phenylalanine

Incorporation of 300 pmol methionine

48 nmol/l polyphenylalani

ne

Incorporation of 197 pmoles [14C ]-leucine

Incorporation of 20

cpm/pmol of methionine

Reaction duration

150 minutes 30 minutes 30-60 minutes 15 minutes 30 minutes 15-60 minutes

- denotes that the information was not accessible to us.

39

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 frame - typically 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

cell-free transcription-translation reactions that last for several hours and can

produce up to 0.8 µM GFPmut3b, which is a ~72x fold-improvement when compared

with a B. subtilis 168 cell-free transcription-translation 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 transcription-translation

systems (production of over 40µM of reporter protein) (Caschera and 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), co-factor

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.

40

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 Pgrac (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 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 characterization 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.

Acknowledgements

We wish to acknowledge the support of the Engineering and Physical Science

Research Council (EPSRC) – [EP/K034359/1; EP/J02175X/1] and that of our

colleagues in the Centre for Synthetic Biology and Innovation (CSynBI) at Imperial

College. We would also like to thank colleagues from The Flowers Consortium,

particularly Professor Colin Harwood, for their support and advice. We also thank

Professor Angelika Gründling for the kind gift of Bacillus subtilis 168.

41

References

Bhatia, S., Densmore, D., 2013. Pigeon: A Design Visualizer for Synthetic Biology. ACS

Synth. Biol. 2, 348–350. doi:10.1021/sb400024s

Burkholder, P.R., Giles, N.H., 1947. Induced biochemical mutations in Bacillus subtilis. Am.

J. Bot. 34, 345–8.

Cai, Q., Hanson, J. a., Steiner, A.R., Tran, C., Masikat, M.R., Chen, R., Zawada, J.F., Sato,

A.K., Hallam, T.J., Yin, G., 2015. A simplified and robust protocol for immunoglobulin

expression in E scherichia coli cell-free protein synthesis systems. Biotechnol. Prog. 31,

823–831. doi:10.1002/btpr.2082

Caschera, F., Noireaux, V., 2015a. Preparation of amino acid mixtures for cell-free

expression systems. doi:10.2144/000114249

Caschera, F., Noireaux, V., 2015b. A cost-effective polyphosphate-based metabolism fuels

an all E. coli cell-free expression system. Metab. Eng. 27, 29–37.

doi:10.1016/j.ymben.2014.10.007

Caschera, F., Noireaux, V., 2014. Synthesis of 2.3 mg/ml of protein with an all Escherichia

coli cell-free transcription-translation system. Biochimie 99, 162–8.

doi:10.1016/j.biochi.2013.11.025

Ceroni, F., Algar, R., Stan, G.-B., Ellis, T., 2015. Quantifying cellular capacity identifies gene

expression designs with reduced burden. Nat. Methods 12, 415–8.

doi:10.1038/nmeth.3339

Chappell, J., Jensen, K., Freemont, P.S., 2013. Validation of an entirely in vitro approach for

rapid prototyping of DNA regulatory elements for synthetic biology. Nucleic Acids Res.

41, 1–11. doi:10.1093/nar/gkt052

Collins, J.J., Gardner, T.S., Cantor, C.R., 2000. Construction of a genetic toggle switch in

Escherichia coli. Nature 403, 339–342. doi:10.1038/35002131

Cutting, S., Vander Horn, P.B., 1990. Genetic Analysis, in: Harwood, C.R., Cutting, S. (Eds.),

Molecular Biological Methods for Bacillus. John Wiley and Sons, Chichester, United

Kingdom, pp. 27–74. doi:10.1016/S0232-4393(11)80231-3

Dudley, Q.M., Karim, A.S., Jewett, M.C., 2015. Cell-free metabolic engineering:

Biomanufacturing beyond the cell. Biotechnol. J. doi:10.1002/biot.201400330

42

Elowitz, M.B., Leibler, S., 2000. A synthetic oscillatory network of transcriptional regulators.

Nature 403, 335–338. doi:10.1038/35002125

Gagoski, D., Polinkovsky, M.E., Mureev, S., Kunert, A., Johnston, W., Gambin, Y.,

Alexandrov, K., 2016. Performance benchmarking of four cell-free protein expression

systems. Biotechnol. Bioeng. 113, 292–300. doi:10.1002/bit.25814

Gan, R., Jewett, M.C., 2014. A combined cell-free transcription-translation system from

Saccharomyces cerevisiae for rapid and robust protein synthe. Biotechnol. J. 9, 641–

651. doi:10.1002/biot.201300545

Garamella, J., Marshall, R., Rustad, M., Noireaux, V., 2016. The All E. coli TX-TL Toolbox

2.0: A Platform for Cell-Free Synthetic Biology. ACS Synth. Biol. 5, 344–55.

doi:10.1021/acssynbio.5b00296

Guan, C., Cui, W., Cheng, J., Zhou, L., Liu, Z., Zhou, Z., 2016. Development of an efficient

autoinducible expression system by promoter engineering in Bacillus subtilis. Microb.

Cell Fact. 15, 66. doi:10.1186/s12934-016-0464-0

Guiziou, S., Sauveplane, V., Chang, H.-J., Cler E, C., Declerck, N., Jules, M., Bonnet, J.,

2016. A part toolbox to tune genetic expression in Bacillus subtilis. Nucleic Acids Res.

1–14. doi:10.1093/nar/gkw624

Hampf, M., Gossen, M., 2006. A protocol for combined Photinus and Renilla luciferase

quantification compatible with protein assays. Anal. Biochem. 356, 94–9.

doi:10.1016/j.ab.2006.04.046

Harwood, C.R., 1992. Bacillus subtilis and its relatives: molecular biological and industrial

workhorses. Trends Biotechnol. 10, 247–56.

Harwood, C.R., Pohl, S., Smith, W., Wipat, A., 2013. Bacillus subtilis. Model Gram-Positive

Synthetic Biology Chassis. Methods Microbiol. 40, 87–117. doi:10.1016/B978-0-12-

417029-2.00004-2

Hodgman, C.E., Jewett, M.C., 2012. Cell-free synthetic biology: Thinking outside the cell.

Metab. Eng. 14, 261–269. doi:10.1016/j.ymben.2011.09.002

Jeong, D.-E., Park, S.-H., Pan, J.-G., Kim, E.-J., Choi, S.-K., 2015. Genome engineering

using a synthetic gene circuit in Bacillus subtilis. Nucleic Acids Res. 43, e42–e42.

doi:10.1093/nar/gku1380

Jewett, M.C., Calhoun, K.A., Voloshin, A., Wuu, J.J., Swartz, J.R., 2008. An integrated cell-

43

free metabolic platform for protein production and synthetic biology. Mol. Syst. Biol. 4.

doi:10.1038/msb.2008.57

Karim, A.S., Jewett, M.C., 2016. A cell-free framework for rapid biosynthetic pathway

prototyping and enzyme discovery. Metab. Eng. 36, 116–126.

doi:10.1016/j.ymben.2016.03.002

Kelly, J.R., Rubin, A.J., Davis, J.H., Ajo-Franklin, C.M., Cumbers, J., Czar, M.J., de Mora, K.,

Glieberman, A.L., Monie, D.D., Endy, D., 2009. Measuring the activity of BioBrick

promoters using an in vivo reference standard. J. Biol. Eng. 3, 4. doi:10.1186/1754-

1611-3-4

Kelwick, R., MacDonald, J.T., Webb, A.J., Freemont, P., 2014. Developments in the Tools

and Methodologies of Synthetic Biology. Front. Bioeng. Biotechnol. 2.

doi:10.3389/fbioe.2014.00060

Legault-Demare, L., Chambliss, G.H., 1974. Natural messenger ribonucleic acid-directed

cell-free protein-synthesizing system of Bacillus subtilis. J. Bacteriol. 120, 1300–7.

Leventhal, J.M., Chambliss, G.H., 1979. DNA-directed cell-free protein-synthesizing system

of Bacillus subtilis. Biochim. Biophys. Acta 564, 162–71.

Lorenz, W.W., McCann, R.O., Longiaru, M., Cormier, M.J., 1991. Isolation and expression of

a cDNA encoding Renilla reniformis luciferase. Proc. Natl. Acad. Sci. U. S. A. 88, 4438–

42. doi:10.1073/pnas.88.10.4438

Matthews, J.C., Hori, K., Cormier, M.J., 1977. Purification and properties of Renilla

reniformis luciferase. Biochemistry 16, 85–91. doi:10.1021/bi00620a014

Moore, S.J., Lai, H.-E., Kelwick, R.J.R., Chee, S.M., Bell, D.J., Polizzi, K.M., Freemont, P.S.,

2016. EcoFlex: A Multifunctional MoClo Kit for E. coli Synthetic Biology. ACS Synth.

Biol. acssynbio.6b00031. doi:10.1021/acssynbio.6b00031

Nes, I.F., Eklund, T., 1983. The effect of parabens on DNA, RNA and protein synthesis in

Escherichia coli and Bacillus subtilis. J. Appl. Bacteriol. 54, 237–42.

Nguyen, H., Phan, T., Schumann, W., 2011. Analysis and application of Bacillus subtilis

sortases to anchor recombinant proteins on the cell wall. AMB Express 1, 22.

doi:10.1186/2191-0855-1-22

Nguyen, H.D., Phan, T.T.P., Schumann, W., 2007. Expression vectors for the rapid

purification of recombinant proteins in Bacillus subtilis. Curr. Microbiol. 55, 89–93.

44

doi:10.1007/s00284-006-0419-5

Niederholtmeyer, H., Sun, Z.Z., Hori, Y., Yeung, E., Verpoorte, A., Murray, R.M., Maerkl,

S.J., 2015. Rapid cell-free forward engineering of novel genetic ring oscillators. Elife 4,

1–5. doi:10.7554/eLife.09771

Nirenberg, M., 2004. Historical review: Deciphering the genetic code – a personal account.

Trends Biochem. Sci. 29, 46–54. doi:10.1016/j.tibs.2003.11.009

Noireaux, V., Bar-Ziv, R., Libchaber, A., 2003. Principles of cell-free genetic circuit

assembly. Proc. Natl. Acad. Sci. 100, 12672–12677. doi:10.1073/pnas.2135496100

Ogawa, A., Namba, Y., Gakumasawa, M., 2016. Rational optimization of amber suppressor

tRNAs toward efficient incorporation of a non-natural amino acid into protein in a

eukaryotic wheat germ extract. Org. Biomol. Chem. 14, 2671–8.

doi:10.1039/c5ob02533h

Okamoto, M., Fukui, S., Kobayashi, Y., 1985. In Vitro Expression of Plasmid pUB110 DNA

with Bacillus subtilis Cell-free Extracts. Agric. Biol. Chem. 49, 1077–1082.

doi:10.1080/00021369.1985.10866864

Pardee, K., Green, A.A., Ferrante, T., Cameron, D.E., DaleyKeyser, A., Yin, P., Collins, J.J.,

2014. Paper-Based Synthetic Gene Networks. Cell 159, 940–954.

doi:10.1016/j.cell.2014.10.004

Patron, N.J., Orzaez, D., Marillonnet, S., Warzecha, H., Matthewman, C., Youles, M.,

Raitskin, O., Leveau, A., Farré, G., Rogers, C., Smith, A., Hibberd, J., Webb, A.A.R.,

Locke, J., Schornack, S., Ajioka, J., Baulcombe, D.C., Zipfel, C., Kamoun, S., Jones,

J.D.G., Kuhn, H., Robatzek, S., Van Esse, H.P., Sanders, D., Oldroyd, G., Martin, C.,

Field, R., O’Connor, S., Fox, S., Wulff, B., Miller, B., Breakspear, A., Radhakrishnan,

G., Delaux, P.-M., Loqué, D., Granell, A., Tissier, A., Shih, P., Brutnell, T.P., Quick,

W.P., Rischer, H., Fraser, P.D., Aharoni, A., Raines, C., South, P.F., Ané, J.-M.,

Hamberger, B.R., Langdale, J., Stougaard, J., Bouwmeester, H., Udvardi, M., Murray,

J.A.H., Ntoukakis, V., Schäfer, P., Denby, K., Edwards, K.J., Osbourn, A., Haseloff, J.,

2015. Standards for plant synthetic biology: a common syntax for exchange of DNA

parts. New Phytol. 208, 13–19. doi:10.1111/nph.13532

Pohl, S., Bhavsar, G., Hulme, J., Bloor, A.E., Misirli, G., Leckenby, M.W., Radford, D.S.,

Smith, W., Wipat, A., Williamson, E.D., Harwood, C.R., Cranenburgh, R.M., 2013.

Proteomic analysis of Bacillus subtilis strains engineered for improved production of

45

heterologous proteins. Proteomics 13, 3298–3308. doi:10.1002/pmic.201300183

Shimizu, Y., Kanamori, T., Ueda, T., 2005. Protein synthesis by pure translation systems.

Methods 36, 299–304. doi:10.1016/j.ymeth.2005.04.006

Shin, J., Noireaux, V., 2012. An E. coli Cell-Free Expression Toolbox: Application to

Synthetic Gene Circuits and Artificial Cells. ACS Synth. Biol. 1, 29–41.

doi:10.1021/sb200016s

Shrestha, P., Holland, T.M., Bundy, B.C., 2012. Streamlined extract preparation for

Escherichia coli-based cell-free protein synthesis by sonication or bead vortex mixing.

Biotechniques 53, 163–74. doi:10.2144/0000113924

Siegal-Gaskins, D., Tuza, Z.A., Kim, J., Noireaux, V., Murray, R.M., 2014. Gene Circuit

Performance Characterization and Resource Usage in a Cell-Free “Breadboard.” ACS

Synth. Biol. 3, 416–425. doi:10.1021/sb400203p

Sullivan, C.J., Pendleton, E.D., Sasmor, H.H., Hicks, W.L., Farnum, J.B., Muto, M., Amendt,

E.M., Schoborg, J.A., Martin, R.W., Clark, L.G., Anderson, M.J., Choudhury, A., Fior,

R., Lo, Y.-H., Griffey, R.H., Chappell, S.A., Jewett, M.C., Mauro, V.P., Dresios, J., 2016.

A cell-free expression and purification process for rapid production of protein biologics.

Biotechnol. J. 11, 238–48. doi:10.1002/biot.201500214

Sun, Z.Z., Hayes, C.A., Shin, J., Caschera, F., Murray, R.M., Noireaux, V., 2013. Protocols

for implementing an Escherichia coli based TX-TL cell-free expression system for

synthetic biology. J. Vis. Exp. e50762. doi:10.3791/50762

Sun, Z.Z., Yeung, E., Hayes, C.A., Noireaux, V., Murray, R.M., 2014. Linear DNA for Rapid

Prototyping of Synthetic Biological Circuits in an Escherichia coli Based TX-TL Cell-

Free System. ACS Synth. Biol. 3, 387–397. doi:10.1021/sb400131a

Sushmita, M.R., Sunil, B., Evan, G., Henry, H., 2015. Method for enhancing recombinant

protein production by cell-free protein expression system. US9040253 (B2).

Takahashi, M.K., Hayes, C. a., Chappell, J., Sun, Z.Z., Murray, R.M., Noireaux, V., Lucks,

J.B., 2015. Characterizing and prototyping genetic networks with cell-free transcription–

translation reactions. Methods 86, 60–72. doi:10.1016/j.ymeth.2015.05.020

Tuza, Z. a., Singhal, V., Jongmin Kim, Murray, R.M., 2013. An in silico modeling toolbox for

rapid prototyping of circuits in a biomolecular &amp;#x201C;breadboard&amp;#x201D;

system, in: 52nd IEEE Conference on Decision and Control. IEEE, pp. 1404–1410.

46

doi:10.1109/CDC.2013.6760079

Webb, A.J., Kelwick, R., Doenhoff, M.J., Kylilis, N., MacDonald, J.T., Wen, K.Y., McKeown,

C., Baldwin, G., Ellis, T., Jensen, K., Freemont, P.S., 2016. A protease-based

biosensor for the detection of schistosome cercariae. Sci. Rep. 6, 24725.

doi:10.1038/srep24725

Westers, L., Westers, H., Quax, W.J., 2004. Bacillus subtilis as cell factory for

pharmaceutical proteins: a biotechnological approach to optimize the host organism.

Biochim. Biophys. Acta - Mol. Cell Res. 1694, 299–310.

doi:10.1016/j.bbamcr.2004.02.011

Zaghloul, T.I., Doi, R.H., 1987. In vitro expression of a Tn9-derived chloramphenicol

acetyltransferase gene fusion by using a Bacillus subtilis system. J. Bacteriol. 169,

1212–6.

Zeigler, D.R., Pragai, Z., Rodriguez, S., Chevreux, B., Muffler, A., Albert, T., Bai, R., Wyss,

M., Perkins, J.B., 2008. The Origins of 168, W23, and Other Bacillus subtilis Legacy

Strains. J. Bacteriol. 190, 6983–6995. doi:10.1128/JB.00722-08

Zhang, C., Tsoi, R., You, L., 2016. Addressing biological uncertainties in engineering gene

circuits. Integr. Biol. 8, 456–464. doi:10.1039/C5IB00275C

Zubay, G., 1973. In vitro synthesis of protein in microbial systems. Annu. Rev. Genet. 7,

267–87. doi:10.1146/annurev.ge.07.120173.001411

47