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

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.

Membrane molecular crowding enhances MreB polymerization to shape synthetic cells from spheres to rods

“…The TXTL system recapitulates physiological conditions and contains all of the nutrients necessary for MreB stability and polymerization, magnesium (∼10 mM) and ATP (∼2 mM) in particular. None of the proteins expressed in this work are present in the lysate, as evidenced recently by mass spectrometry analysis of this cell-free system (30). All of the performed reactions contained the same ingredients, except for the plasmids that were changed based on the proteins synthesized.…”

Methodologies for preparation of prokaryotic extracts for cell-free expression systems

“…While extremely useful as a standard across labs due to ease of measurement, the approach hides potentially large and complex underlying differences between methods [ 35 ], ignores the fraction of non-functional protein produced [ 151 ], and could poorly predict performance for some applications. The recent application of proteomics [ 112 , [152] , [153] , [154] ] and metabolomics [ 155 ] tools are important advances to better understand the underlying differences between extracts and CFE systems more generally.…”

Cell-Free Approach for Non-canonical Amino Acids Incorporation Into Polypeptides

“…It involves cell disruption followed by a run-off incubation procedure that frees the ribosomes and degrades the mRNAs, several high-speed centrifugations that removes non-essential proteins. Approximately 500–1,000 proteins were identified in E. coli S30 lysate (A19 and BL21 Rosetta2) (Foshag et al, 2018 ; Garenne et al, 2019 ). These represent 20–40% of the E. coli proteome (Iwasaki et al, 2010 ; Schmidt et al, 2016 ).…”

“…However, the S30 system contains other factors, such as nucleases, RNases, and proteases, that negatively affect the translation productivity (Foshag et al, 2018 ; Garenne et al, 2019 ). The presence of endo- and exo- nucleases causes rapid decay of linear PCR templates.…”