10.1021/sb400003y.s001
Roberta Lentini
Roberta
Lentini
Michele Forlin
Michele
Forlin
Laura Martini
Laura
Martini
Cristina Del Bianco
Cristina Del
Bianco
Amy C. Spencer
Amy C.
Spencer
Domenica Torino
Domenica
Torino
Sheref S. Mansy
Sheref S.
Mansy
Fluorescent Proteins and <i>in Vitro</i> Genetic Organization for Cell-Free Synthetic Biology
American Chemical Society
2015
coli translation machinery
protein
T 7 RNA polymerase
ratiometric fluorescence assay
restriction sites
ribosome binding sites
ability
sequence composition influences
spacing
Vitro Genetic Organization
device
data
expression
2015-12-16 23:35:01
Journal contribution
https://acs.figshare.com/articles/journal_contribution/Fluorescent_Proteins_and_i_in_Vitro_i_Genetic_Organization_for_Cell_Free_Synthetic_Biology/2025807
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 <i>in
vitro</i> transcription-translation reactions with a minimal
system consisting of T7 RNA polymerase and <i>E. coli</i> translation machinery, i.e., the PUREsystem. The data were used
to construct a ratiometric fluorescence assay to quantify the effect
of genetic organization on <i>in vitro</i> 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 <i>in vitro</i>.