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Communication pathways between artificial cells can be used to build artificial consortia for a variety of purposes, including bioremediation and biomedical applications. Communication based on small molecules is desirable so that complex transport paths across membranes can be avoided. To explore the feasibility of setting up molecular communication between two different kinds of artificial cells, liposomes carrying an E. coli based transcription/translation machinery were incubated with proteinosomes. Like liposomes, proteinosomes are able to create compartments in aqueous solution. E. coli transcription/translation machinery allows for the regulation of a desired function at a genetic level, while proteinosomes allow for facile localization of a specific function (through enzymatic activity) on the membrane of the compartment. The communication pathway that we designed involves the release of a small molecule from the liposomes through the synthesis of a pore-forming protein regulated at a genetic level. The released chemical signal is sensed by enzymes on the surface of the proteinosome and then further processed by enzymes within the lumen of the proteinosome. Dario Cecchi,1 Dora Tang,2 Stephen Mann,2 Sheref S. Mansy1 1 CIBIO, University of Trento, via Sommarive 9 38123 Povo (TN) Italy 2 Centre for Protolife Research, School of Chemistry, University of Bristol, Bristol BS8 1TS, United Kingdom 0 10 20 30 40 50 60 70 80 Luminescence fold increase SAM [mM ] 0.3 0 0 0 0.3 Acetyl-CoA [mM ] 0.01 0 0 0 0.01 3OC6 [mM ] 0 0 0.01 0 0 LuxI [mM ] 4.6 4.6 0 0 0 Hexanoyl-ACP N-(3-Oxohexanoyl)-L-homoserine lactone (3OC6) A proteinosomes made of glucose oxidase (GOx) carrying horse radish peroxidase (HRP) in its lumen is able to carry out a reaction that converts amplex red into the fluorescent molecule resorufin. Increasing amounts of glucose correlate with increasing fluorescence, measured at a plate reader (exc.: 560 nm; em.: 584 nm). The communication molecule glucose (green hexagon) is released through αhemolysin (αHL) pores from liposomes and detected by proteinosomes. POPC:cholesterol (1:2) liposomes encapsulate a cell-free transcription/translation (TX/TL) mix and a plasmid encoding αHL under the control of a genetic switch. Proteinosomes carry out an enzymatic reaction using glucose as a substrate and converting a non fluorescent molecule into a fluorescent dye to allow for direct measurement. S. aureus α-hemolysin is under the control of a V. fischeri quorum sensing regulatory mechanism. Briefly, LuxR activates transcription of αHL in the presence of N-(3Oxohexanoyl)-L-homoserine lactone (3OC6). The efficacy of the genetic switch was checked by the release of the self-quenching fluorophore calcein from liposomes. Excitation and emission wavelengths were 485 nm and 515 nm, respectively. Similarly, glucose release was monitored with a coupled enzymatic reaction by following the reduction of NAD+ to NADH by fluorescence (exc.: 339 nm; em.: 450 nm). Systems running in the opposite direction are also being built where vesicle-based systems are able to sense proteinosomes. S-adenosylmethionine 0 200 400 600 800 1000 1200 1400 1600 LuxR/αHL - 3OC6 LuxR/αHL + 3OC6 LuxR/βGal - 3OC6 LuxR/βGal + 3OC6 0.6% Triton X-100 0.6 mg/ml αHL 5% glycerol buffer 5% glycerol Fluorescence (A. U.) LuxR 3OC6 Ptet luxR Plux LuxR/αHL αHL LuxR 3OC6 Ptet luxR Plux LuxR/αHL FFL LuxI GOx HRP O2 H2O2 Amplex Red Resorufin Gluconic acid Glucose Purified acyl homoserine lactone synthase (LuxI) within proteinosomes produce enough 3OC6 to induce gene expression inside liposomes. The activity of the enzyme was tested in a TX/TL reaction together with the reporter plasmid carrying a firefly luciferase gene under the control of the V. fischeri quorum sensing system. The system is efficient even in absence of the precursor molecules, suggesting their presence in the E. coli extract. 0 100 200 300 400 500 600 700 Buffer 70 mM glucose HEPES 0.3% Triton X-100 DC119A - 3OC6 DC119A + 3OC6 Buffer 0.3 % Triton X-100 DC119A - 3OC6 DC119A + 3OC6 Controls Vesicles with buffer Vesicles with 100 mM glucose Fluorescence (A. U.) G6PDH ATP ADP HK Glucose Glucose 6-phosphate 6-phospho-D-glucono-1,5-lactone NAD+ NADH
