IGEM:IMPERIAL/2007/Projects/In-Veso

In-Veso Gene Expression: Introduction

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

The vesicle chassis is an addition to the Registry of Standard Biological Parts. In these pages we detail the techniques and characteristics of its use.

The specification, design, modelling, implementation, and testing cycle is applied to the characterisation of the chassis. The specifications outline the characteristics of the chassis being sought, and the quantities that will be varied in experiments. The design elaborates how these characteristics will be obtained, including experimental protocols, and experiment schedules. Some characteristics are obtained from literature, and these are not covered in the design. In modelling, we seek to refine the experimental design, identify expected points of interest in the results, and provide a more abstracted description of the chassis itself. The section on implementation covers the actual experiments and their results. Finally, in testing and validation, the results are analysed and compared to the modelling and specifications.

The In Vitro Gene Expression System

The in vitro expression of proteins in cell-free extracts is an important tool for molecular biologists and has a variety of applications. The use of in vitro translation systems can have advantages over in vivo gene expression when the over-expressed product is toxic to the host cell, when the product is insoluble or forms inclusion bodies, or when the protein undergoes rapid proteolytic degradation by intracellular proteases. [1] The big advantage of the in vitro approach in our projects, is that it enables the detection of biofilm on medical devices and the detection of spoilage in meat, without actually having to place E.coli in contact with the medical devices or the meat.

There are two approaches to in vitro protein synthesis based on the starting genetic material: RNA or DNA. Standard translation systems use RNA as a template; whereas coupled transcription-translation systems start with DNA templates, which are transcribed into RNA then translated. DNA templates for coupled transcription-translation systems can be easily generated by PCR. The most frequently used cell-free systems consist of extracts from rabbit reticulocytes, wheat germ and E.coli. For the purposes of our projects, the E.coli system looks more attractive than the eukaryotic systems.

Unlike eukaryotic systems where transcription and translation occur sequentially, in E. coli systems, coupled transcription and translation occur simultaneously in the same tube under the same reaction conditions. This bacterial system gives efficient expression of either prokaryotic or eukaryotic gene products in a short amount of time. Capping of eukaryotic RNA is not required. Use of E.coli extract also eliminates cross-reactivity or other problems associated with endogenous proteins in eukaryotic lysates. In addition, the E. coli S30 extract system allows expression from DNA vectors containing natural E. coli promoter sequences. [2] A more streamlined E.coli S12 extract has also been developed as an improvement to the S30 extract. [3]

Transcription can be performed by T7, SP16 or E.coli RNA polymerases. The E.coli RNA polymerase is the preferred choice for our projects because the reporter genes to be expressed are downstream of E.coli promoter sequences (such as pLux).

The In Veso Gene Expression System

Austin "Veso" Powers - special imaging techniques reveal his lipid bi-layer membrane structure.

The in veso gene expression system is somewhat like building an artificial cell. The E.coli in vitro gene expression system is encapsulated in a phospholipid vesicle. Unilamellar vesicles containing the E.coli cell extracts are produced in an oil–extract emulsion. The vesicles are then transferred into a feeding solution that contains ribonucleotides and amino acids to form a bilayer. To create a selective permeability for nutients, the α-hemolysin pore protein from Staphylococcus aureus is expressed inside the vesicle. Coupled transcription:translation of plasmid genes can thus be compartmentalized in the vesicles. [4]

No other known biological or synthetic barrier gives as many possibilities and as much flexibility to functionalize and establish controllable exchanges between inside and outside. Cell-free expression in emulsion droplets brings expression to the scale of the cell but does not allow continuous expression because exchanges with the solvent phase are difficult. [5]

Comparison of In vitro and In veso

1. One order of magnitude increase in protein production and duration of expression in in veso is obtained when compared to batch mode.
2. Batch mode can last less than a day, as expression stops because of the decrease of the adenylate energy charge. Synthetic phospholipid vesicles can last up to four days.
3. Only bacteriophage polymerases can be used in veso, while E.coli polymerases can be used in vitro.
4. There will be osmotic pressure buildup when making vesicles. This can be counteracted by addition of polymers or inert proteins into the feeding solution.

Progress

References

1. In Vitro Translation: The Basics (Ambion)

2. Methods for producing proteins by using cell-free protein synthesis systems

3. 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 |
4. Noireaux V and Libchaber A. A vesicle bioreactor as a step toward an artificial cell assembly. Proc Natl Acad Sci U S A. 2004 Dec 21;101(51):17669-74. DOI:10.1073/pnas.0408236101 |
5. 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 |