title: Cell-Free Translation Is More Variable than Transcription.
abstract: Although RNA synthesis can be reliably controlled with different T7 transcriptional promoters during cell-free gene expression with the PURE system, protein synthesis remains largely unaffected. To better control protein levels, we investigated a series of ribosome binding sites (RBSs). Although RBS strength did strongly affect protein synthesis, the RBS sequence could explain less than half of the variability of the data. Protein expression was found to depend on other factors besides the strength of the RBS, including the GC content of the coding sequence. The complexity of protein synthesis in comparison to RNA synthesis was observed by the higher degree of variability associated with protein expression. This variability was also observed in an E. coli cell extract-based system. However, the coefficient of variation was larger with E. coli RNA polymerase than with T7 RNA polymerase, consistent with the increased complexity of E. coli RNA polymerase.
authors.affiliation: Center for Integrative Biology (CIBIO), University of Trento , via Sommarive 9, 38123 Povo TN, Italy.
authors.forename: Fabio
authors.initials: F
authors.lastname: Chizzolini
authors.affiliation: Center for Integrative Biology (CIBIO), University of Trento , via Sommarive 9, 38123 Povo TN, Italy.
authors.forename: Michele
authors.initials: M
authors.lastname: Forlin
authors.affiliation: Center for Integrative Biology (CIBIO), University of Trento , via Sommarive 9, 38123 Povo TN, Italy.
authors.forename: Noël
authors.initials: N
authors.lastname: Yeh Martín
authors.affiliation: Center for Integrative Biology (CIBIO), University of Trento , via Sommarive 9, 38123 Povo TN, Italy.
authors.forename: Giuliano
authors.initials: G
authors.lastname: Berloffa
authors.affiliation: Center for Integrative Biology (CIBIO), University of Trento , via Sommarive 9, 38123 Povo TN, Italy.
authors.forename: Dario
authors.initials: D
authors.lastname: Cecchi
authors.affiliation: Center for Integrative Biology (CIBIO), University of Trento , via Sommarive 9, 38123 Povo TN, Italy.
authors.forename: Sheref S
authors.identifier: 0000-0003-2382-198X
authors.initials: SS
authors.lastname: Mansy
mesh_terms: D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D001665:Binding Sites; D002474:Cell-Free System / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D038761:Photobleaching; D014176:Protein Biosynthesis; D011506:Proteins / Q000378:metabolism; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D059370:RNA Folding; D012270:Ribosomes / Q000378:metabolism; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000737:chemistry / Q000378:metabolism; D000097573:Red Fluorescent Protein
chemical_list: D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D012321:DNA-Directed RNA Polymerases; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D011506:Proteins; D012313:RNA; D014764:Viral Proteins; D000097573:Red Fluorescent Protein; C062210:bacteriophage T7 RNA polymerase
keywords: IVTT; PURE system; artificial cell; cell-free synthetic biology; transcription-translation