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      Engineering genetic circuit interactions within and between synthetic minimal cells

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          Abstract

          Genetic circuits and reaction cascades are of great importance for synthetic biology, biochemistry, and bioengineering. An open question is how to maximize the modularity of their design to enable the integration of different reaction networks and to optimize their scalability and flexibility. One option is encapsulation within liposomes which enables chemical reactions to proceed in well-isolated environments. Here we adapt liposome encapsulation to enable the modular, controlled compartmentalization of genetic circuits and cascades. We demonstrate that it is possible to engineer genetic circuit-containing synthetic minimal cells (synells) to contain multiple-part genetic cascades, and that these cascades can be controlled by external signals as well as inter-liposomal communication without cross-talk. We also show that liposomes containing different cascades can be fused in a controlled way so that the products of incompatible reactions can be brought together. Synells thus enable more modular creation of synthetic biology cascades, an essential step towards their ultimate programmability.

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          Most cited references53

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          A vesicle bioreactor as a step toward an artificial cell assembly.

          An Escherichia coli cell-free expression system is encapsulated in a phospholipid vesicle to build a cell-like bioreactor. Large unilamellar vesicles containing extracts are produced in an oil-extract emulsion. To form a bilayer the vesicles are transferred into a feeding solution that contains ribonucleotides and amino acids. Transcription-translation of plasmid genes is isolated in the vesicles. Whereas in bulk solution expression of enhanced GFP stops after 2 h, inside the vesicle permeability of the membrane to the feeding solution prolongs the expression for up to 5 h. To solve the energy and material limitations and increase the capacity of the reactor, the alpha-hemolysin pore protein from Staphylococcus aureus is expressed inside the vesicle to create a selective permeability for nutrients. The reactor can then sustain expression for up to 4 days with a protein production of 30 muM after 4 days. Oxygen diffusion and osmotic pressure are critical parameters to maintain expression and avoid vesicle burst.
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            Prebiotic systems chemistry: new perspectives for the origins of life.

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              Experimental models of primitive cellular compartments: encapsulation, growth, and division.

              The clay montmorillonite is known to catalyze the polymerization of RNA from activated ribonucleotides. Here we report that montmorillonite accelerates the spontaneous conversion of fatty acid micelles into vesicles. Clay particles often become encapsulated in these vesicles, thus providing a pathway for the prebiotic encapsulation of catalytically active surfaces within membrane vesicles. In addition, RNA adsorbed to clay can be encapsulated within vesicles. Once formed, such vesicles can grow by incorporating fatty acid supplied as micelles and can divide without dilution of their contents by extrusion through small pores. These processes mediate vesicle replication through cycles of growth and division. The formation, growth, and division of the earliest cells may have occurred in response to similar interactions with mineral particles and inputs of material and energy.
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                Author and article information

                Journal
                101499734
                35773
                Nat Chem
                Nat Chem
                Nature chemistry
                1755-4330
                1755-4349
                15 September 2016
                14 November 2016
                May 2017
                14 May 2017
                : 9
                : 5
                : 431-439
                Affiliations
                [1 ]Media Lab, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA
                [2 ]Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA
                [3 ]McGovern Institute for Brain Research, Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA
                Author notes
                [* ]Correspondence and request for materials should be addressed to E.S.B. esb@ 123456media.mit.edu
                [¥]

                These authors contributed equally to this work

                Article
                NIHMS816451
                10.1038/nchem.2644
                5407321
                28430194
                f6a52d4c-3dff-4cfd-b03c-7dd00e91f995

                Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms

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                Chemistry
                Chemistry

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