title: The All E. coli TX-TL Toolbox 2.0: A Platform for Cell-Free Synthetic Biology.
abstract: We report on and provide a detailed characterization of the performance and properties of a recently developed, all Escherichia coli, cell-free transcription and translation system. Gene expression is entirely based on the endogenous translation components and transcription machinery provided by an E. coli cytoplasmic extract, thus expanding the repertoire of regulatory parts to hundreds of elements. We use a powerful metabolism for ATP regeneration to achieve more than 2 mg/mL of protein synthesis in batch mode reactions, and more than 6 mg/mL in semicontinuous mode. While the strength of cell-free expression is increased by a factor of 3 on average, the output signal of simple gene circuits and the synthesis of entire bacteriophages are increased by orders of magnitude compared to previous results. Messenger RNAs and protein degradation, respectively tuned using E. coli MazF interferase and ClpXP AAA+ proteases, are characterized over a much wider range of rates than the first version of the cell-free toolbox. This system is a highly versatile cell-free platform to construct complex biological systems through the execution of DNA programs composed of synthetic and natural bacterial regulatory parts.
authors.affiliation: School of Physics and Astronomy, University of Minnesota , 115 Union Street SE, Minneapolis, Minnesota 55455, United States.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Garamella
authors.affiliation: School of Physics and Astronomy, University of Minnesota , 115 Union Street SE, Minneapolis, Minnesota 55455, United States.
authors.forename: Ryan
authors.initials: R
authors.lastname: Marshall
authors.affiliation: School of Physics and Astronomy, University of Minnesota , 115 Union Street SE, Minneapolis, Minnesota 55455, United States.
authors.forename: Mark
authors.initials: M
authors.lastname: Rustad
authors.affiliation: School of Physics and Astronomy, University of Minnesota , 115 Union Street SE, Minneapolis, Minnesota 55455, United States.
authors.forename: Vincent
authors.initials: V
authors.lastname: Noireaux
mesh_terms: D001435:Bacteriophages / Q000235:genetics / Q000502:physiology; D002474:Cell-Free System; D004268:DNA-Binding Proteins / Q000378:metabolism; D049071:Endopeptidase Clp / Q000378:metabolism; D004722:Endoribonucleases / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism* / Q000821:virology; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism; D058615:Synthetic Biology; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C422955:MazF protein, E coli; D012333:RNA, Messenger; D014157:Transcription Factors; D049452:Green Fluorescent Proteins; D004722:Endoribonucleases; C483573:ClpXP protease, E coli; D049071:Endopeptidase Clp
keywords: biosynthesis; cell-free transcription−translation; gene circuits prototyping; minimal cell; self-assembly