Refinement of OnePot PURE and Crude Ribosome Production for Reproducible Cell-free Protein Synthesis

Summary

We present an updated protocol for the production of the OnePot 'Protein synthesis Using Recombinant Elements' (PURE) system and crude ribosomes for cell-free protein synthesis, focusing on controlling cell growth to minimize batch-to-batch variations.

Abstract

Recent advances have enabled the Protein synthesis Using Recombinant Elements (PURE) cell-free system to be produced in individual laboratories economically and with reduced labor burden. However, the preparation of the 36 protein components and ribosome, which make up PURE, is still a complex undertaking, with much scope for variation and error. We present a detailed and updated procedure to manufacture PURE based on the recently published OnePot protocol, which involves regulating a number of key steps, in particular, the inoculation of cultures using optical density (OD)-normalized glycerol stocks, careful monitoring of cell growth, and controlling final glycerol concentrations. Using this approach, we report reproducible preparations of PURE from multiple biological replicates and users. This protocol serves as a reference and starting point for other laboratories to manufacture and troubleshoot their own PURE systems, thus increasing democratization of the many applications that PURE enables, such as the bottom-up construction of biomolecular systems and the development of synthetic cells.

Introduction

Cell-free protein synthesis (CFPS) has matured as a technology for diverse applications, including biomanufacturing, biosensing and diagnostics, prototyping of genetic and metabolic parts and pathways, rapid protein engineering, and education^1,2. CFPS systems using purified components, originally developed as the 'Protein synthesis Using Recombinant Elements' (PURE) system³, offer a number of advantages over their lysate-based alternatives. PURE systems have a defined composition, consisting of 36 protein factors, the 70S E. coli ribosome, and a defined mixture of chemi....

Protocol

The protocol is divided into three parts. For the sake of conciseness and clarity, we emphasize the important changes specific to this work, and therefore, details which are the same as those existing in other publications are referenced.

Preliminary work only needs to be carried out once. The four sections involve preparing glycerol stocks and starter plates for the PURE proteins, as well as verifying and measuring expression times of the strains. The use of starter plates removes variability associated with direct inoculation from scraped glycerol stocks. It is crucial to ensure this work is done thoroughly before proceeding to the manufa....

Results

The workflow for the PURE and ribosome preparations is shown in Figure 1, and a recommended timetable for the work is shown in Table 1. Typically, reactions are monitored through the production of fluorescent proteins such as GFP or mCherry, and RFU readings can be converted to concentrations by calibration¹¹. However, CFPS yields depend on the specific protein being produced, and so it is important to optimize the sys.......

Discussion

We have presented a detailed procedure that has allowed us to obtain reproducible PURE systems over multiple batches and users in the same lab. There are a number of critical steps required to achieve this.

The first important step is to ensure the integrity of the 36 PURE strains: in addition to sequencing all plasmids, it is essential to carry out an expression trial to verify all strains are overexpressing correctly, as the loss of a single strain can abolish CFPS activity. We have observed.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2x YT	Sigma	Y2377-250G
amino acids	Sigma	LAA21-1KT
ammonium acetate RNAse-free	Thermo Scientific	AM9070G
ammonium chloride	Sigma	09718-1KG
ampicillin	Sigma	A8351-5G
BL21(DE3) competent E. coli	NEB	C2527H
bottle, centrifuge (Nalgene)	Thermo Scientific	3120-9500	polypropylene co-polymer bottle
bottle, glass (Duran) 250 mL, 500 mL, 1 L, 2 L	VWR	215-1593, 215-1594, 215-1595, 215-1596
Bradford assay, Quick Start 1x Dye Reagent	Bio-Rad	5000205
centrifuge	Thermo Scientific	Heraeus Megafuge 16R
centrifuge, micro	Eppendorf	5424R
chromatography column	Bio-Rad	7321010
creatine phosphate	Sigma	27920
cuvettes, semi micro 1.6 mL	Fisherbrand	FB55147
di-sodium hydrogen orthophophosphate dihydrate	Fisher Chemical	S/4450/60
dialysis cassette, Slide-A-Lyzer G3, 2 kDa MWCO, 15 mL	Thermo Scientific	A52962
dipotassium hydrogen orthophosphate anhydrous	Fisher Chemical	P/5240/60
DTT No-Weigh	Thermo Scientific	A39255
filter unit, centrifugal 3kDa MWCO Amicon Ultra 15	EMD Millipore	UFC900324
flask, 500 mL	VWR	214-1133
flask, baffled 2.5 L	Tunair	SS-5013
folinic acid	Sigma	F7878-100MG
glycerol	Sigma	G5516-1L
HCl	Fisherbrand	H/1200/PB17
HEPES buffer, 1 M solution pH 7.3	Thermo Scientific	BP299
imidazole	Sigma	I2399-500G
incubator	Eppendorf	New Brunswick Innova® 42
IPTG	Thermo Scientific	R0392
KOH pellets	Thermo Scientific	P1767-500G
L-Glutamic acid hemimagnesium salt tetrahydrate	Sigma	49605-250G
L-Glutamic acid monopotassium salt monohydrate	Sigma	49601-500G
magnesium acetate tetrahydrate	Sigma	M5661-50G
magnesium chloride	Fluka	63020-1L
magnetic stirring setup	Stuart	US152
Nanodrop	Thermo Scientific	Nanodrop 2000
NEBExpress Ni-NTA Mag Beads	NEB	S1423S
Parafilm	Sigma	P7543-1EA
pipette, multichannel Eppendorf Research Plus 30-300 µL	Eppendorf	3125000052
plate adhesive seal for plate reader	ExcelScientific	STR-SEAL-PLT (Sterile)
plate breathable seal	Starlab	E2796-3005
plate foil seal	ExcelScientific	FCS-25
plate reader	BioTek	Synergy H1
plate shaker	Allsheng	MBI100-4A
plate, 384 well	Thermo Scientific	242764
plate, 96 deep well 1 mL	Thermo Scientific	736-0601
plate, 96 deep well 2 mL	Sarstedt	82.1972.002
plate, 96 well PCR	Sarstedt	721,978
plate, optical 96 well	Thermo Scientific	10057581
potassium chloride	Sigma	P5405-500G
potassium dihydrogen orthophosphate	Fisher Chemical	P/4800/53
protein extraction (BugBuster) reagent	EMD Millipore	70584-3
protein purification resin (cOmplete)	Roche	5893682001
rotisserie shaker	Starlab	RM-Multi 1
sodium chloride	Fisher Chemical	S/3105/70
sonication probe, FisherBrand 15-50 mL	Thermo Scientific	12931181
sonicator	Fisher Scientific	FB120
spectrophotometer	Jenway	Genova Plus
spermidine	Sigma	S2626-1G
TCEP 0.5 M	Supelco	646547
Thermo Fisher NTP, Tris buffered	Thermo Scientific	R1481
tRNA from E. coli MRE600	Roche	10109541001
tryptone	Formedium	TRP02
tube, 0.2 mL PCR grade	Sarstedt	72,737,002
tube, bijoux Sterilin 7 mL polystyrene	Thermo Scientific	129A
tube, conical (Falcon) 15 mL	Sarstedt	62.554.502
tube, conical (Falcon) 50 mL	Sarstedt	62.547.254
tube, microcentrifuge (Eppendorf) 1.5 mL	Sarstedt	72.690.001
tube, microcentrifuge (Eppendorf) 2 mL	Sarstedt	72.695.700
tube, S100 centrifugation	Beckman-Coulter	355619
tube, S30 centrifugation	Thermo Scientific	3118-0050
ultracentrifuge	Beckman-Coulter	Optima XPN-100
ultracentrifuge rotor	Beckman-Coulter	70Ti fixed angle rotor
UltraPure DNase/RNase-Free Distilled Water	Thermo Scientific	10977049
vortexer	VWR	444-2791
yeast extract	Oxoid	LP0021T