BBa_K260018 1 BBa_K260018 Cell free Chassis: Mass production of aqueous vesicles in oil phase 2009-10-20T11:00:00Z 2015-05-08T01:11:42Z does not apply. part contains no sequence Method to mass produce vesicles of uniform shape and volume Vesicle characterization Content of the vesicles can be chosen freely. Environment inside vesicles is aquious. Vesicle shape: spherical Vesicle diameter: 15 ??m Vesicle volume: ~12 pl Vesicle population has a very narrow distribution in size. Production efficiency: 5-15 vesicles per second Vesicle stability: not fully characterized, depends on surfactant used, at least 2 hours; fusion of vesicles observed after >24 hours Optical properties: transparent in brightfield microscopy; for observation of fluorescence the surfactant used should be non-fluorescent Vesicle isolation through output hole. Vesicle content is released when vesicles transfered to aqueous Protocol You will need a silicone waver with the engraved microfluidic system, and a micropump system Setup of the microfluidic system The microfluidic system consists of a flow chamber made of Polydimethylsiloxane (PDMS) and a pump system that controls the flow rates of the various liquids into the chamber. Droplets are created within a defined space in the chamber and are propagated along a grid that allows containment and imaging. Two types of chambers have been used, differing in the geometry of the space where droplets were produced. One featured T-junction, and the other a V-junction. Production of flow chambers: * mix PDMS and curing agent in 10:1 ratio * degas and pour on wafer with etched microstructures * polymerize on heat plate at 150 ??C for 30 min * add unpolymerized PDMS mixture to points on microstructure where microtube inlets are to be pierced * polymerize on heat plate at 150 ??C for 30 minutes * remove polymerized PDMS from wafer, cut to fit onto glass cover slide (24 x 60 mm), and use clean needles (0.8 mm) or laser cutter (Trotec Speedy 100TM) to pierce tube inlets * ionize PDMS and glass slide in plasma chamber for 30 sec to make it reactive * align PDMS on glass slide and seal * seal irreversibly by heating on plate at 60??C for 6 hours The pumping system (ceDOSYS SP-4) allows control of syringes filled with aqueous material and mineral oil treated with surfactant(1% Span 80), respectively. The syringes access the chamber via the tubing inlets. Two inlets are used to pump in material in the aqueous phase; the remaining one is used for the oil phase. The flow rates of the syringes are controlled via a ceDOSYS user interface software. Control via pump system: * two syringes are loaded with 1ml each of material in the aqueous phase; during the first trial, distilled water * another is filled with a 1ml solution of 0.5% span 80 in oil * use flow rate on ceDOSYS interface to flood the chamber first with oil phase * gradually introduce aqueous phase and modify rates of both phases until the shear stress breaks the aqueous phase into droplets at the T- or V- junctions in the respective chambers. false false _356_ 0 4384 9 No part sequence true does not apply. part contains no sequence false Deepikaa Menon and Tomasz Sadowski igem2sbol 1 iGEM to SBOL conversion Conversion of the iGEM parts registry to SBOL2.1 Chris J. Myers James Alastair McLaughlin 2017-03-06T15:00:00.000Z