Chassis/Cell-Free Systems

Revision as of 22:32, 20 October 2007 by Cheuk Ka Tong (Talk | contribs) (→‎Introduction)

Cell-Free Systems

Introduction

Cell-Free Systems (CFS) involve the in-vitro expression of genes into proteins. These systems can serve as a compatible chassis for the various parts and devices from the Registry of Standard Biological Parts.

Coupled transcription-translation systems usually combine a bacteriophage RNA polymerase and promoter with eukaryotic or prokaryotic extracts. In addition, the PURE system has been developed as a reconstituted CFS for synthesizing proteins using recombinant elements.

Advantages and disadvantages of CFS

Advantages
Disadvantages
Non-infectious because of non-proliferative nature Short expression lifespan since system cannot replicate
Process is quick and simple requiring only preparation of cell extract and feeding solution and subsequent addition of DNA template Expensive because of the constant need for nutrient and energy supply
Quality control can be achieved easily using modified reaction conditions such as addition of accessory elements or inhibitory factors Less characterization and experience of use in the laboratories compared to E. coli


Specifications for CFS characterization
The following are several parameters that will help us understand the advantages and disadvantages associated with a particular chassis housing the gene expression machinery.

Properties
Definitions
Rise time Measure of time from start of reaction to the point when expression rate first reaches the steady state value.
Peak time Measure of time from start of reaction to the point when expression rate reaches the maximum value.
Settling time Measure of time from start of reaction to the point when expression rate reaches the steady state and does not escape from it for a prolonged period of time.
Stability of synthesized protein Measure of the half-life of a given protein (e.g. GFP) in the chassis.
Total expression capacity Measure of the total expression of a chassis for a given DNA construct template. This should take into account the degradation of synthesized protein.
Expression lifespan Measure of time that expression occurs for a given DNA construct template until protein degradation overrides protein synthesis.

Cell-Free Systems investigated



References

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