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

In-Veso Gene Expression: Design

• Introduction
• Specifications
• Design
• Modelling
• Implementation
• Testing/Validation
• Notes
• References

Summary

Characterisation Stages - this image needs updating.

Development of the vesicle chassis will proceed in two paths, in parallel.
Path 1: In-vitro expression system

• Establishes a platform for comparison of techniques
• Tests whether constructs and components are working

Path 2: In-veso chassis development

• 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

Simple procedures for the construction of a robust and cost-effective cell-free protein synthesis system [1]

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:

Formation of vesicles [2]

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.

References

1. Kim TW, Keum JW, Oh IS, Choi CY, Park CG, and Kim DM. Simple procedures for the construction of a robust and cost-effective cell-free protein synthesis system. J Biotechnol. 2006 Dec 1;126(4):554-61. DOI:10.1016/j.jbiotec.2006.05.014 |
2. Noireaux V, Bar-Ziv R, Godefroy J, Salman H, and Libchaber A. Toward an artificial cell based on gene expression in vesicles. Phys Biol. 2005 Sep 15;2(3):P1-8. DOI:10.1088/1478-3975/2/3/P01 |
3. Pellinen T, Huovinen T, and Karp M. A cell-free biosensor for the detection of transcriptional inducers using firefly luciferase as a reporter. Anal Biochem. 2004 Jul 1;330(1):52-7. DOI:10.1016/j.ab.2004.03.064 |