Preparation of Amino Acid Mixtures for Cell-Free Expression Systems

Caschera, Filippo; Noireaux, Vincent

doi:10.2144/000114249

Cited by 47 publications

(42 citation statements)

References 16 publications

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“…The energy buffer for ATP regeneration is composed of the following: 50 mM Hepes pH 8, 1.5 mM ATP and GTP, 0.9 mM CTP and UTP, 0.2 mg/mL tRNA, 0.26 mM coenzyme A, 0.33 mM NAD, 0.75 mM cAMP, 0.068 mM folinic acid, 1 mM spermidine, 30 mM 3‐PGA, 20–40 mM maltodextrin. A typical cell‐free reaction is composed of 33% (v/v) of E. coli lysate, the other 66% of the reaction volume includes the energy mix, the amino acid solution (3 mM of each of the 20 amino acids) and plasmids. Mg‐glutamate, K‐glutamate, and PEG8000 concentrations were adjusted to 5 mM, 60 mM and 2%, respectively .…”

Section: Methodsmentioning

confidence: 99%

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

Garenne

Beisel

Noireaux

2019

Rapid Comm Mass Spectrometry

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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.

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Section: Methodsmentioning

confidence: 99%

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

Garenne

Beisel

Noireaux

2019

Rapid Comm Mass Spectrometry

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“…CFPS reactions were performed as described by Oza et al 47 Some reagents can be pre-mixed, aliquoted, and stored at -20 ºC (*pre-mix, † amino acid mix prepared as described 94 ; ‡ salt mix at pH 7 with KOH) and the final reaction should be at pH 6.5-7. CFPS reactions were initiated by thoroughly mixing the cell extract, incubated for 17-20 hours at 30 ºC, and performed in 15, 30, or 50 uL reactions.…”

Section: Phosphoserine Incorporation Of Recombinant Proteins Using Cementioning

confidence: 99%

Pptc7 is an essential phosphatase for promoting mammalian mitochondrial metabolism and biogenesis

Niemi

Wilson

Overmyer

et al. 2018

Preprint

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20functional relevance of these modifications are largely unclear. Nonetheless, mitochondria 21 possess multiple resident phosphatases, suggesting that protein dephosphorylation may be 22 broadly important for mitochondrial activities. To explore this, we deleted the poorly characterized 23 matrix phosphatase Pptc7 from mice using CRISPR-Cas9 technology. Strikingly, Pptc7 -/mice 24 exhibited marked hypoketotic hypoglycemia, elevated acylcarnitines, and lactic acidosis, and died 25 soon after birth. Pptc7 -/tissues had significantly diminished mitochondrial size and protein content 26 despite normal transcript levels, but consistently elevated phosphorylation on select mitochondrial 27 proteins. These putative Pptc7 substrates include the protein translocase complex subunit 28 Timm50, whose phosphorylation reduced import activity. We further find that phosphorylation in 29 or near the mitochondrial targeting sequences of multiple proteins can disrupt their import rates 30 and matrix processing. Overall, our data define Pptc7 as a protein phosphatase essential for 31 proper mitochondrial function and biogenesis during the extrauterine transition. 33 34Mitochondria are multifaceted organelles required for metabolic and signaling processes within 35 almost every eukaryotic cell type 1 . Beyond their production of ATP through oxidative 36 phosphorylation, mitochondria play key roles in macromolecular biosynthesis, ion homeostasis, 37 redox signaling, and apoptotic cell deathactivities that must be calibrated to changing cellular 38 needs. Recent investigations suggest that these and other mitochondrial functions may be 39 affected by post-translational modifications (PTMs), such as phosphorylation 2,3 and acylation 4,5 . 40These PTMs are found on hundreds of mitochondrial proteins 6,7 , and can alter enzyme function 6 , 41 complex assembly 8 , and metabolic flux 9,10 . However, other studies suggest that these 42 mitochondrial PTMs can arise non-enzymatically 11,12 and are often found at low stoichiometry 2,13-43 15 , calling into question whether and to what extent these modifications exert regulatory functions. 44Although much remains to be established about the overall nature and importance of 45 reversible phosphorylation in mitochondria, it is clear that these organelles possess a number of 46 resident phosphatases [16][17][18] . For example, it has long been known that the pyruvate 47 dehydrogenase 9 and branched chain ketoacid dehydrogenase complexes 19 include bound 48 phosphatases (and kinases) that regulate their activities. However, beyond these, there exist 49 other poorly characterized mitochondrial proteins that possess known or predicted protein 50 phosphatase domains [16][17][18] , suggesting that protein dephosphorylation may be of more widespread 51 importance in mitochondria than is currently appreciated. To begin exploring this, we recently 52 analyzed three S. cerevisiae mitochondrial phosphatase deletion strains (∆ptc5, ∆ptc6, and 53 ∆ptc7) 18 and identified phenotypes and putative substrates uni...

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“…Basic Cell-Free reaction consists of a crude cell extract from E. coli (which contains endogenous transcriptiontranslation machinery, mRNA and protein degradation enzymes plus all the soluble proteins), buffer and DNA. The amino acid mix was prepared following the protocol from [9]. All the experiments shown here are performed using the same batch of extract preparation to avoid variation between different batches.…”

Section: Cell-free Reaction Conditionsmentioning

confidence: 99%

Translation Inhibition and Resource Balance in the Cell-Free Gene Expression System

Nagaraj

Greene

Sengupta

et al. 2017

Preprint

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Quantifying the effect of vital resources on transcription and translation helps to understand the degree to which the concentration of each resource must be regulated for achieving homeostasis. Utilizing the synthetic transcription-translation (TX-TL) system, we study the impact of nucleotide triphosphates (NTPs) and magnesium (Mg 2+ ), on gene expression. Recent observations of the counterintuitive phenomenon of suppression of gene expression at high NTP concentrations have led to the speculation that such suppression is due to the consumption of resources by transcription, hence leaving fewer resources for translation. In this work, we investigate an alternative hypothesis: direct suppression of the translation rate via stoichiometric mismatch in necessary reagents. We observe NTP-dependent suppression even in the early phase of gene expression, contradicting the resource limitation argument. To further decouple the contributions of transcription and translation, we performed gene expression experiments with purified mRNA. Simultaneously monitoring mRNA and protein abundances allowed us to extract a time-dependent translation rate. Measuring translation rates for different Mg 2+ and NTP concentrations, we observe a complex resource dependence. We demonstrate that translation is the rate-limiting process that is directly inhibited by high NTP concentrations. Additional Mg 2+ can partially reverse this inhibition. In several experiments, we observe two maxima of the translation rate viewed as a function of both Mg 2+ and NTP concentration, which can be explained in terms of an NTP-independent effect on the ribosome complex and an NTP-Mg2 + titration effect. The non-trivial compensatory effects of abundance of different vital resources signals the presence of complex regulatory mechanisms to achieve optimal gene expression.

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Preparation of Amino Acid Mixtures for Cell-Free Expression Systems

Cited by 47 publications

References 16 publications

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

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

Pptc7 is an essential phosphatase for promoting mammalian mitochondrial metabolism and biogenesis

Translation Inhibition and Resource Balance in the Cell-Free Gene Expression System

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