Fluorescent proteins and in vitro genetic organization for cell-free synthetic biology.

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.

[1]  Yutetsu Kuruma,et al.  Biosynthesis of proteins inside liposomes. , 2010, Methods in molecular biology.

[2]  T. D. Schneider,et al.  Anatomy of Escherichia coli ribosome binding sites. , 2001, Journal of molecular biology.

[3]  L. Martini,et al.  Cell-like systems with riboswitch controlled gene expression. , 2011, Chemical communications.

[4]  Pasquale Stano,et al.  Achievements and open questions in the self-reproduction of vesicles and synthetic minimal cells. , 2010, Chemical communications.

[5]  V. Noireaux,et al.  An E. coli cell-free expression toolbox: application to synthetic gene circuits and artificial cells. , 2012, ACS synthetic biology.

[6]  T. Tenson,et al.  Translation initiation region sequence preferences in Escherichia coli , 2007, BMC Molecular Biology.

[7]  Thomas F. Knight,et al.  BioBrick vectors from BioBrick parts , 2015 .

[8]  Petra Schwille,et al.  Towards a bottom-up reconstitution of bacterial cell division. , 2012, Trends in cell biology.

[9]  Hiroshi Kita,et al.  Constructing partial models of cells. , 2010, Cold Spring Harbor perspectives in biology.

[10]  Takuya Ueda,et al.  Cell-free translation reconstituted with purified components , 2001, Nature Biotechnology.

[11]  Michael S. Waterman,et al.  RNA Secondary Structure , 1995 .

[12]  D. Endy,et al.  Refactoring bacteriophage T 7 , 2006 .

[13]  T. Yomo,et al.  Kinetic analysis of aptazyme-regulated gene expression in a cell-free translation system: modeling of ligand-dependent and -independent expression. , 2012, RNA.

[14]  R. Kwok.  Five hard truths for synthetic biology , 2010, Nature.

[15]  Vincent Noireaux,et al.  Development of an artificial cell, from self-organization to computation and self-reproduction , 2011 .

[16]  M. Inouye,et al.  Downstream box: a hidden translational enhancer , 1998, Molecular microbiology.

[17]  M Bjerknes,et al.  Determination of the optimal aligned spacing between the Shine-Dalgarno sequence and the translation initiation codon of Escherichia coli mRNAs. , 1994, Nucleic acids research.

[18]  C. S. Devine,et al.  The T7 phage gene 10 leader RNA, a ribosome-binding site that dramatically enhances the expression of foreign genes in Escherichia coli. , 1988, Gene.

[19]  David K. Karig,et al.  Expression optimization and synthetic gene networks in cell-free systems , 2011, Nucleic acids research.

[20]  S. Mansy,et al.  Cellular imitations. , 2012, Current opinion in chemical biology.

[21]  I. V. Boni,et al.  Ribosome-messenger recognition: mRNA target sites for ribosomal protein S1 , 1991, Nucleic Acids Res..

[22]  Y. Mechulam,et al.  Translation Initiation , 2020, Definitions.

[23]  J. van Duin,et al.  Secondary structure of the ribosome binding site determines translational efficiency: a quantitative analysis. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[24]  M. Smit,et al.  Secondary structure of the ribosome binding site determines translational efficiency: a quantitative analysis. , 1990 .

[25]  R. Breaker,et al.  Regulation of bacterial gene expression by riboswitches. , 2005, Annual review of microbiology.

[26]  Kazufumi Hosoda,et al.  Replication of Genetic Information with Self‐Encoded Replicase in Liposomes , 2008, ChemBioChem.

[27]  George M Church,et al.  Towards synthesis of a minimal cell , 2006, Molecular systems biology.

[28]  Nathan C Shaner,et al.  A guide to choosing fluorescent proteins , 2005, Nature Methods.

[29]  G. Stormo,et al.  Translation initiation in Escherichia coli: sequences within the ribosome‐binding site , 1992, Molecular microbiology.

[30]  Michael C Jewett,et al.  Cell-free biology: exploiting the interface between synthetic biology and synthetic chemistry. , 2012, Current opinion in biotechnology.

[31]  M. Kozak.  Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes , 1986, Cell.

[32]  D. Endy,et al.  Refactoring bacteriophage T7 , 2005, Molecular systems biology.

[33]  F. Hartl,et al.  De novo folding of GFP fusion proteins: high efficiency in eukaryotes but not in bacteria. , 2005, Journal of molecular biology.

[34]  T. Yomo,et al.  Synthesis of functional proteins within liposomes. , 2010, Methods in molecular biology.

[35]  Drew Endy,et al.  Engineering BioBrick vectors from BioBrick parts , 2008, Journal of Biological Engineering.

[36]  Andrew Buchanan,et al.  Coping with complexity: Machine learning optimization of cell‐free protein synthesis , 2011, Biotechnology and Bioengineering.

[37]  Najaf A. Shah,et al.  Broad-Specificity mRNA–rRNA Complementarity in Efficient Protein Translation , 2012, PLoS genetics.