Characterization of the all-E. coli transcription-translation system myTXTL by mass spectrometry

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16 Scopus citations

Abstract

Rationale: Cell-free transcription-translation (TXTL) is becoming a popular technology to prototype and engineer biological systems outside living organisms. TXTL relies commonly on a cytoplasmic extract that provides the molecular components necessary to recapitulate gene expression in vitro, where most of the available systems are derived from E. coli. The proteinic and enzymatic composition of lysates, however, is typically unknown. In this work, we analyzed by mass spectrometry the molecular constituents of the all-E. coli TXTL platform myTXTL prepared from the E. coli strain BL21 Rosetta2. Methods: Standard TXTL reactions were assembled and executed for 10–12 hours at 29°C. In addition to a no-DNA control, four DNA programs were executed in separate reactions to synthesize the reporter protein deGFP as well as the phages MS2, phix174 and T7. The reactions were treated according to standard procedures (trypsin treatment, cleaning) before performing liquid chromatography/mass spectrometry (LC/MS). Data analysis was performed using Sequest and protein identification using Scaffold. Results: A total of 500–800 proteins were identified by LC/MS in the blank reactions. We organized the most abundant protein sets into several categories pertaining, in particular, to transcription, translation and ATP regeneration. The synthesis of deGFP was easily measured. The major structural proteins that compose the three phages MS2, phix174 and T7 were also identified. Conclusions: Mass spectrometry is a practical tool to characterize biochemical solutions as complex as a cell-free TXTL reaction and to determine the presence of synthesized proteins. The data presented demonstrate that the composition of TXTL based on lysates can be used to validate some underlying molecular mechanisms implicated in cell-free protein synthesis. The composition of the lysate shows significant differences with respect to similar studies on other E. coli strains.

Original languageEnglish (US)
Pages (from-to)1036-1048
Number of pages13
JournalRapid Communications in Mass Spectrometry
Volume33
Issue number11
DOIs
StatePublished - May 15 2019

Bibliographical note

Funding Information:
The authors thank LeeAnn Higgins, Todd W. Markowski and Alan Zimmerman from the Center for Mass Spectrometry and Proteomics, University of Minnesota, for technical help and assistance in data analysis. Funding was received from the Defense Advanced Research Projects Agency, contract HR0011‐16‐C‐01‐34 (to C.L.B. and V.N.) and from the Human Frontier Science Program, research grant RGP0037/2015 (to V.N.).

Publisher Copyright:
© 2019 John Wiley & Sons, Ltd.

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