Rapid and Scalable Preparation of Bacterial Lysates for Cell-Free Gene Expression

Cell-free
gene expression systems are emerging as an important
platform for a diverse range of synthetic biology and biotechnology
applications, including production of robust field-ready biosensors.
Here, we combine programmed cellular autolysis with a freeze–thaw
or freeze-dry cycle to create a practical, reproducible, and a labor-
and cost-effective approach for rapid production of bacterial lysates
for cell-free gene expression. Using this method, robust and highly
active bacterial cell lysates can be produced without specialized
equipment at a wide range of scales, making cell-free gene expression
easily and broadly accessible. Moreover, live autolysis strain can
be freeze-dried directly and subsequently lysed upon rehydration to
produce active lysate. We demonstrate the utility of autolysates for
synthetic biology by regulating protein production and degradation,
implementing quorum sensing, and showing quantitative protection of
linear DNA templates by GamS protein. To allow versatile and sensitive
β-galactosidase (LacZ) based readout we produce autolysates
with no detectable background LacZ activity and use them to produce
sensitive mercury­(II) biosensors with LacZ-mediated colorimetric and
fluorescent outputs. The autolysis approach can facilitate wider adoption
of cell-free technology for cell-free gene expression as well as other
synthetic biology and biotechnology applications, such as metabolic
engineering, natural product biosynthesis, or proteomics.