@santannapisa.it
The Biorobotics Institute
Scopus Publications
T-Y. Dora Tang, Dario Cecchi, Giorgio Fracasso, Davide Accardi, Angelique Coutable-Pennarun, Sheref S. Mansy, Adam W. Perriman, J. L. Ross Anderson, and Stephen Mann
American Chemical Society (ACS)
A gene-directed chemical communication pathway between synthetic protocell signaling transmitters (lipid vesicles) and receivers (proteinosomes) was designed, built and tested using a bottom-up modular approach comprising small molecule transcriptional control, cell-free gene expression, porin-directed efflux, substrate signaling, and enzyme cascade-mediated processing.
Fabio Chizzolini, Michele Forlin, Noël Yeh Martín, Giuliano Berloffa, Dario Cecchi, and Sheref S. Mansy
American Chemical Society (ACS)
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.
Samir Ounzain, Rudi Micheletti, Carme Arnan, Isabelle Plaisance, Dario Cecchi, Blanche Schroen, Ferran Reverter, Michael Alexanian, Christine Gonzales, Shi Yan Ng,et al.
Elsevier BV
Dario Cecchi and Sheref S. Mansy
Springer International Publishing
Fabio Chizzolini, Michele Forlin, Dario Cecchi, and Sheref S. Mansy
American Chemical Society (ACS)
The cell-free transcription-translation of multiple proteins typically exploits genes placed behind strong transcriptional promoters that reside on separate pieces of DNA so that protein levels can be easily controlled by changing DNA template concentration. However, such systems are not amenable to the construction of artificial cells with a synthetic genome. Herein, we evaluated the activity of a series of T7 transcriptional promoters by monitoring the fluorescence arising from a genetically encoded Spinach aptamer. Subsequently the influences of transcriptional promoter strength on fluorescent protein synthesis from one, two, and three gene operons were assessed. It was found that transcriptional promoter strength was more effective at controlling RNA synthesis than protein synthesis in vitro with the PURE system. Conversely, the gene position within the operon strongly influenced protein synthesis but not RNA synthesis.
Roberta Lentini, Silvia Perez Santero, Fabio Chizzolini, Dario Cecchi, Jason Fontana, Marta Marchioretto, Cristina Del Bianco, Jessica L. Terrell, Amy C. Spencer, Laura Martini,et al.
Springer Science and Business Media LLC
AbstractPrevious efforts to control cellular behaviour have largely relied upon various forms of genetic engineering. Once the genetic content of a living cell is modified, the behaviour of that cell typically changes as well. However, other methods of cellular control are possible. All cells sense and respond to their environment. Therefore, artificial, non-living cellular mimics could be engineered to activate or repress already existing natural sensory pathways of living cells through chemical communication. Here we describe the construction of such a system. The artificial cells expand the senses of Escherichia coli by translating a chemical message that E. coli cannot sense on its own to a molecule that activates a natural cellular response. This methodology could open new opportunities in engineering cellular behaviour without exploiting genetically modified organisms.