Quantitative modeling of transcription and translation of an all-E. coli cell-free system. 2019

Ryan Marshall, and Vincent Noireaux
School of Physics and Astronomy, University of Minnesota, 115 Union Street SE, Minneapolis, MN, 55455, USA. marsh752@umn.edu.

Cell-free transcription-translation (TXTL) is expanding as a polyvalent experimental platform to engineer biological systems outside living organisms. As the number of TXTL applications and users is rapidly growing, some aspects of this technology could be better characterized to provide a broader description of its basic working mechanisms. In particular, developing simple quantitative biophysical models that grasp the different regimes of in vitro gene expression, using relevant kinetic constants and concentrations of molecular components, remains insufficiently examined. In this work, we present an ODE (Ordinary Differential Equation)-based model of the expression of a reporter gene in an all E. coli TXTL that we apply to a set of regulatory elements spanning several orders of magnitude in strengths, far beyond the T7 standard system used in most of the TXTL platforms. Several key biochemical constants are experimentally determined through fluorescence assays. The robustness of the model is tested against the experimental parameters, and limitations of TXTL resources are described. We establish quantitative references between the performance of E. coli and synthetic promoters and ribosome binding sites. The model and the data should be useful for the TXTL community interested either in gene network engineering or in biomanufacturing beyond the conventional platforms relying on phage transcription.

UI MeSH Term Description Entries
D008954 Models, Biological Theoretical representations that simulate the behavior or activity of biological processes or diseases. For disease models in living animals, DISEASE MODELS, ANIMAL is available. Biological models include the use of mathematical equations, computers, and other electronic equipment. Biological Model,Biological Models,Model, Biological,Models, Biologic,Biologic Model,Biologic Models,Model, Biologic
D002474 Cell-Free System A fractionated cell extract that maintains a biological function. A subcellular fraction isolated by ultracentrifugation or other separation techniques must first be isolated so that a process can be studied free from all of the complex side reactions that occur in a cell. The cell-free system is therefore widely used in cell biology. (From Alberts et al., Molecular Biology of the Cell, 2d ed, p166) Cellfree System,Cell Free System,Cell-Free Systems,Cellfree Systems,System, Cell-Free,System, Cellfree,Systems, Cell-Free,Systems, Cellfree
D004926 Escherichia coli A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc. Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
D000465 Algorithms A procedure consisting of a sequence of algebraic formulas and/or logical steps to calculate or determine a given task. Algorithm
D014158 Transcription, Genetic The biosynthesis of RNA carried out on a template of DNA. The biosynthesis of DNA from an RNA template is called REVERSE TRANSCRIPTION. Genetic Transcription
D014176 Protein Biosynthesis The biosynthesis of PEPTIDES and PROTEINS on RIBOSOMES, directed by MESSENGER RNA, via TRANSFER RNA that is charged with standard proteinogenic AMINO ACIDS. Genetic Translation,Peptide Biosynthesis, Ribosomal,Protein Translation,Translation, Genetic,Protein Biosynthesis, Ribosomal,Protein Synthesis, Ribosomal,Ribosomal Peptide Biosynthesis,mRNA Translation,Biosynthesis, Protein,Biosynthesis, Ribosomal Peptide,Biosynthesis, Ribosomal Protein,Genetic Translations,Ribosomal Protein Biosynthesis,Ribosomal Protein Synthesis,Synthesis, Ribosomal Protein,Translation, Protein,Translation, mRNA,mRNA Translations
D015964 Gene Expression Regulation, Bacterial Any of the processes by which cytoplasmic or intercellular factors influence the differential control of gene action in bacteria. Bacterial Gene Expression Regulation,Regulation of Gene Expression, Bacterial,Regulation, Gene Expression, Bacterial

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