title: Fluorescent proteins and in vitro genetic organization for cell-free synthetic biology.
abstract: To facilitate the construction of cell-free genetic devices, we evaluated the ability of 17 different fluorescent proteins to give easily detectable fluorescence signals in real-time from in vitro transcription-translation reactions with a minimal system consisting of T7 RNA polymerase and E. coli translation machinery, i.e., the PUREsystem. The data were used to construct a ratiometric fluorescence assay to quantify the effect of genetic organization on in vitro expression levels. Synthetic operons with varied spacing and sequence composition between two genes that coded for fluorescent proteins were then assembled. The resulting data indicated which restriction sites and where the restriction sites should be placed in order to build genetic devices in a manner that does not interfere with protein expression. Other simple design rules were identified, such as the spacing and sequence composition influences of regions upstream and downstream of ribosome binding sites and the ability of non-AUG start codons to function in vitro.
authors: CIBIO, University of Trento , via delle Regole 101, 38123 Mattarello (TN), Italy.
authors: Roberta
authors: R
authors: Lentini
authors: Michele
authors: M
authors: Forlin
authors: Laura
authors: L
authors: Martini
authors: Cristina
authors: C
authors: Del Bianco
authors: Amy C
authors: AC
authors: Spencer
authors: Domenica
authors: D
authors: Torino
authors: Sheref S
authors: SS
authors: Mansy
mesh_terms: D002474:Cell-Free System; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005453:Fluorescence; D015870:Gene Expression; D005821:Genetic Techniques; D016015:Logistic Models; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D014176:Protein Biosynthesis; D012270:Ribosomes / Q000235:genetics / Q000378:metabolism; D058615:Synthetic Biology / Q000379:methods*; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000235:genetics
chemical_list: D008164:Luminescent Proteins; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases