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TRANSCRIPT
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: [email protected] 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
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