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

Abstract

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.

Simple, Functional, Inexpensive Cell Extract forin vitroPrototyping of Proteins with Disulfide Bonds

“…21,32 Previous literature has shown that increasing extract concentration can lead to increased expression rates at the cost of a reduction in total protein produced. 47 In agreement with literature, concentrating the extract led to a sharp decrease in levels of Gluc luminescence as can be seen in Fig 2C. Surprisingly, subsequent dilutions outperformed the standard, non-diluted extract.…”

Design of a transcriptional biosensor for the portable, on-demand detection of cyanuric acid

“…This work represents the first implementation of an LTTR in a bacterial extract, expanding the range of aTFs that have been shown to be functional in freeze-dried, cell-free systems beyond the wellcharacterized transcriptional regulators like MerR and TetR. [28][29][30] In addition, to our knowledge, our best design represents the most sensitive (low detection limit) CYA biosensor reported in the literature. More broadly, this work uncovers design rules for both E. coli whole-cell and cell-free biosensors composed of non-native transcriptional regulators.…”

Engineering Cell‐Free Protein Synthesis for High‐Yield Production and Human Serum Activity Assessment of Asparaginase: Toward On‐Demand Treatment of Acute Lymphoblastic Leukemia

“…The procedure is also low cost: one test well of ASNase assay CFPS reaction costs about $0.14, for a total cost of <$2.00 for each test if a complete standard is run in parallel. Furthermore, streamlined cell extract preparation procedures are making CFPS technology increasingly available to prospective laboratories . Quantifying ASNase in 290 mOsM HEPES/PEG‐400 buffer suggests that the assay will be robust in solutions used to stabilize ASNase (Figure S4, Supporting Information).…”

“…Furthermore, streamlined cell extract preparation procedures are making CFPS technology increasingly available to prospective laboratories. [38] Quantifying ASNase in 290 mOsM HEPES/PEG-400 buffer suggests that the assay will be robust in solutions used to stabilize ASNase ( Figure S4, Supporting Information). The quantification of ASNase activity in human serum is especially promising because: 1) this is the first time to our knowledge that active CFPS has been reported in the presence of human serum, and 2) detecting ASNase activity in the presence of 44% serum suggests that this assay could help improve ALL treatment by monitoring patient serum ASNase activity.…”