Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Deepikaa Menon and Tomasz Sadowski
Date created: 2009-10-20 11:00:00
Date modified: 2015-05-08 01:11:42
Cell free Chassis: Mass production of aqueous vesicles in oil phase
| Types | DnaRegion |
| Roles | sequence_feature Other |
Description
Method to mass produce vesicles of uniform shape and volumeVesicle 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.