IGEM:IMPERIAL/2007/Projects/In-Veso/Design

=In-Veso Gene Expression: Design=

 Introduction Specifications Design Modelling Implementation Testing/Validation Notes References </ul> <br style="clear:both">

Summary
Development of the vesicle chassis will proceed in two paths, in parallel. Path 1: In-vitro expression system Path 2: In-veso chassis development
 * Establishes a platform for comparison of techniques
 * Tests whether constructs and components are working
 * Master the process of vesicle formation
 * Characterise vesicles as a viable chassis

For both paths, there are two preparatory steps that need to be carried out - preparing the DNA constructs to be tested, and preparing the chassis (solution or vesicles). Between the two, the only step that varies is the preparation of the chassis. Further, the only difference between the in-vitro and in-veso preparations is the additional step of creating vesicles. Both chassis will be using the same cell extracts. The DNA constructs and experiments will be the same for both stages.

Path 1: In-vitro expression system


There are two choices for making cell extract from E.coli. One of them is S30, and the other is the S12 method, which is a more rapid and cost-effective preparation. The cell extracts can also be obtained from a manufacturer.

We have protocols for boths extracts, to be chosen depending on the E.coli strain that will be used:
 * S30 E.coli Extract Protocol
 * S12 E.coli Extract Protocol

A single batch of E.coli extract will be prepared for use in all experiments.

Commercially available E.coli S30 extract from Promega and Ambion:
 * E.coli S30 extract system for circular DNA:
 * L1020
 * 278 GBP for 30 rxn
 * E.coli T7 S30 extract system for circular DNA:
 * 1130
 * 278 GBP for 30 rxn
 * E.coli S30 extract system for linear templates:
 * 1030
 * 278 GBP for 30 rxn
 * PROTEINscript® II T7 linked transcription:translation kits for circular or linear DNA:
 * AM1281/6
 * 320 GBP for 40 rxn
 * 100 GMP for 10 rxn

Both the pure E.coli and T7-pol commercial S30 extracts are prepared by modifications of the method described by Zubay (1973, 1980) from an E. coli strain B deficient in OmpT endoproteinase and lon protease activity.

ATP regenerating system
For preparation of the cell extract, we would want to use a cell-free protein synthesis system that is capable of using pyruvate as an energy source to produce high yields of protein. The Cytomim system, synthesizes chloramphenicol acetyltransferase (CAT) for up to 6 h in a batch reaction to yield 700 g/mL of protein. It provides a stable energy supply for protein expression without phosphate accumulation, pH change, exogenous enzyme addition, or the need for expensive high-energy phosphate compounds.

Path 2: In-veso chassis development
The formation of vesicles is integral to our project. Here is a short time-line of the process, and its integration with the rest of the project:



A summary of the mineral oil method The mineral oil vesicle peraparation method has 3 stages, regardless of composition or equipment:


 * 1) Preparation of lipid-oil suspension
 * 2) Emulsification of vesicle contents
 * 3) Bi-layer formation through sedimentation

Each step will vary in time and technique. The current protocol requires 2 hours and an overnight incubation for step 1, and at least 3hrs each for steps 2 and 3.

Right now, we are working on finalising the vesicle preparation protocol:


 * Vesicle Preparation Protocol

Materials Required
R9379 Rhodamine B isothiocyanate–Dextran from Sigma link

Information RITC-Dextran average mol wt ~70,000 MDL number MFCD00132176

Fluorescein-12-dUTP link Fluorescein-12-2'-deoxy-uridine-5'-triphosphate (Fluorescein is bound to deoxyuridine triphosphate via an amide linkage. ) Serial no.: 11373242910 Amount: 25 nmol (25 µl)

Use fluorescein-12-dUTP to add a nonradioactive label to DNA. The labeled DNA can easily and safely be detected (e.g ., either directly or with an enzyme-conjugated anti-fluorescein). Formula: C39H37N4O21P3Li4 Molecular weight: Mr = 1018.4 (fluorescein-12-dUTP-Li4)

P9511 3-sn-Phosphatidic acid sodium salt from egg yolk lecithin from Sigma link ≥98%  Synonym 1,2-Diacyl-sn-glycero-3-phosphate sodium salt L-α-Phosphatidic acid sodium salt PA MDL number MFCD00063023

Prepared from L-α-phosphatidylcholine by hydrolysis with cabbage phospholipase D.