Rapid, Affordable, and Uncomplicated Production of Bacterial Cell-free Lysate

Summary

This protocol describes a rapid and simple method to produce bacterial lysate for cell-free gene expression, using an engineered strain of Escherichia coli and requiring only standard laboratory equipment.

Abstract

Cell-free gene expression offers the power of biology without the complications of a living organism. Although many such gene expression systems exist, most are quite expensive to buy and/or require special equipment and finely honed expertise to produce effectively. This protocol describes a method to produce bacterial cell-free lysate that supports high levels of gene expression, using only standard laboratory equipment and requiring minimal processing. The method uses an Escherichia coli strain producing an endolysin that does not affect growth, but which efficiently lyses a harvested cell pellet following a simple freeze-thaw cycle. The only further processing required is a brief incubation followed by centrifugation to clear the autolysate of cellular debris. Dynamic gene circuits can be achieved through heterologous expression of the ClpX protease in the cells before harvesting. An E. coli strain lacking the lacZ gene can be used for high-sensitivity, cell-free biosensing applications using a colorimetric or fluorescent readout. The entire protocol requires as few as 8-9 hours, with only 1-2 hours of hands-on labor from inoculation to completion. By reducing the cost and time to obtain cell-free lysate, this method should increase the affordability of cell-free gene expression for various applications.

Introduction

Gene expression in cell-free lysates has several advantages over using live cells^1,2,3,4. Lysates can be easily modified biochemically and used in conditions that could be detrimental to or impossible to achieve in live cells. Gene expression circuits do not have to contend or compete with host biological processes, and testing new genetic circuits is as simple as adding DNA. For these reasons, cell-free gene expression has found various applications, from biosensors^5,6 to rapidly pr....

Protocol

1. Prepare media and buffers.

1. Prepare 2xYTPG medium.
   1. Mix 62 g 2xYT powder, 5.99 g potassium phosphate monobasic, 13.93 g potassium phosphate dibasic, and deionized water to 2 L.
   2. Autoclave on liquid cycle with an exposure time of 30 min¹⁴.
   3. To 400 mL of 2xYTP media from 1.1.2, add 7.2 g D-glucose (dextrose) and mix until dissolved.
   4. Filter-sterilize through a 0.2 µm filter.
2. Prepare S30A buffer.
   1. Mix Tris-HCl (pH 7.7, 50 mM final concentration), potassium glutamate (60 mM final), and magnesium glutamate (14 mM final).
   2. Adjust the pH to 7.7 usi....

Results

Representative results can be observed by using autolysate to express GFP from a constitutively expressing plasmid, here pBEST-OR2-OR1-Pr-UTR1-deGFP-T500, and recording a time course of GFP fluorescence in a plate reader (Figure 1B). A dilution series of plasmid DNA found strong expression even at 1 nM DNA. Compared to a commercially available lysate, the autolysate can produce a greater total yield and achieved a greater maximum production rate, calculated as the time derivative of the GFP .......

Discussion

The protocol described here yields highly active bacterial lysate for cell-free gene expression. The key is to use cells carrying the plasmid pAD-LyseR, which expresses the lambda phage endolysin cytosolically. These cells are potentiated to lyse themselves upon permeabilization of the inner membrane, allowing the endolysin access to the cell wall, which the method achieves through a simple freeze-thaw cycle. Because the cells effectively lyse themselves, the product is referred to as autolysate. After the cells have lys.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2xYT media	EMD Millipore	4.85008	or equivalent
3-PGA	Sigma Aldrich	P8877	or equivalent
Amicon Ultra-15 centrifugal filter unit, 3 kDa cutoff	Millipore Sigma	UFC900308	optional, can be used to concentrate lysate, select concentrator capacity appropriate for the volume to be concentrated
ampicillin	Sigma Aldrich	A0166-5G	or carbenicillin, a more stable variant
ATP	Sigma Aldrich	A8937	or equivalent
cAMP	Sigma Aldrich	A9501	or equivalent
CoA	Sigma Aldrich	C4282	or equivalent
CTP	United States Biosciences	14121	or equivalent
D-glucose (dextrose)	Fisher Scientific	AAA1749603	or equivalent
dithiothreitol (DTT)	Sigma Aldrich	D0632-1G	or equivalent
E. coli BL21-Gold (DE3) carrying pAD-LyseR	Addgene	99244
E. coli BL21-Gold (DE3) ΔlacZ carrying pAD-LyseR	Addgene	99245
Folinic acid	Sigma Aldrich	F7878	or equivalent
GTP	United States Biosciences	16800	or equivalent
HEPES	Sigma Aldrich	H3375-25G	or equivalent
LB media	Fisher Scientific	DF0446075	or equivalent
magnesium glutamate	Sigma Aldrich	49605-250G	or equivalent
NAD	Sigma Aldrich	N6522	or equivalent
potassium glutamate	Sigma Aldrich	G1501-100G	or equivalent
potassium hydroxide (KOH)	Sigma Aldrich	221473-25G	for adjusting pH
potassium phosphate dibasic	Fisher Scientific	BP363-500	or equivalent
potassium phosphate monobasic	Fisher Scientific	BP362-500	or equivalent
Spermidine	Sigma Aldrich	85558	or equivalent
Tris-HCl	Fisher Scientific	9310500GM	or equivalent
tRNA mix	Roche	10109541001	or equivalent
UTP	United States Biosciences	23160	or equivalent