Difference between revisions of "Chassis/Cell-Free Systems"

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==Introduction==
 
==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 [https://parts.igem.org/wiki/index.php/Main_Page Registry of Standard Biological Parts].
 
'''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 [https://parts.igem.org/wiki/index.php/Main_Page Registry of Standard Biological Parts].
  
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The Central Dogma describes gene expression in terms of two essential processes - the transcription of DNA into messenger RNA (mRNA) and the translation of mRNA into polypeptides. Not only do CFS house the molecular machinery necessary for transcription and translation, they are also optimized for these two processes. Coupled transcription-translation systems usually combine a bacteriophage RNA polymerase and promoter with eukaryotic or prokaryotic extracts rich in ribosomes, transfer RNAs and aminoacyl-tRNA transferase enzymes. Buffers are also added to maintain the appropriate magnesium and salt concentrations required for efficient translation.
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The Central Dogma <cite>1</cite> describes gene expression in terms of two essential processes - the transcription of DNA into messenger RNA (mRNA) and the translation of mRNA into polypeptides. Not only do CFS house the molecular machinery necessary for transcription and translation, they are also optimized for these two processes. Coupled transcription-translation systems usually combine a bacteriophage RNA polymerase and promoter with eukaryotic or prokaryotic extracts rich in ribosomes, transfer RNAs and aminoacyl-tRNA transferase enzymes. Buffers are also added to maintain the appropriate magnesium and salt concentrations required for efficient translation.
  
  
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In addition, the PURE system has been developed as a reconstituted CFS for synthesizing proteins using recombinant elements <cite>1</cite>.
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In addition, the PURE system has been developed as a reconstituted CFS for synthesizing proteins using recombinant elements <cite>3</cite>.
 
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Revision as of 22:59, 23 October 2007

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.

The Central Dogma 1 describes gene expression in terms of two essential processes - the transcription of DNA into messenger RNA (mRNA) and the translation of mRNA into polypeptides. Not only do CFS house the molecular machinery necessary for transcription and translation, they are also optimized for these two processes. Coupled transcription-translation systems usually combine a bacteriophage RNA polymerase and promoter with eukaryotic or prokaryotic extracts rich in ribosomes, transfer RNAs and aminoacyl-tRNA transferase enzymes. Buffers are also added to maintain the appropriate magnesium and salt concentrations required for efficient translation.


In addition, the PURE system has been developed as a reconstituted CFS for synthesizing proteins using recombinant elements 3.

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
Several parameters are identified to help us understand the advantages and disadvantages associated with using a particular chassis.

Properties
Definitions
Optimum temperature Temperature at which expression rate reaches a maximum value.
Peak time Measure of time from start of reaction to the point when expression rate reaches the maximum value.
Product stability Measure of the half-life of a given protein (e.g. GFP) synthesized in the chassis.
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 modelling

  • To understand better some of the specificities of this new chassis we have built a basic model.
  • Our study investigates the mechanism of constitutive gene expression, inside a cell-free system with limited resources.
  • Read more about our model.

Cell-Free Systems investigated

Please click on each system for its individual characterization.

Homemade E. coli S30 Commercial E. coli S30 Commercial E. coli T7 S30 Vesicle-encapsulation


Different compartmentalization strategies

Batch-mode Transcription-translation reaction is carried out in bulk solution.
Continuous-exchange Transcription-translation reaction is separated from feeding solution by a dialysis membrane.
Vesicle-encapsulated The reaction is separated from feeding solution by a phospholipid bilayer. More reliable exchange of materials is established by inserting a non-specific pore protein into the phospholipid bilayer.2


DNA constructs selected for CFS characterization

  • A simple constitutive gene expression device that reguires an E. coli RNA polymerase BBa_I13522
  • A simple constitutive gene expression device that requires a T7 bacteriophage RNA polymerase BBa_R0085
  • An inducible gene expression device that is well-characterized in the registry BBa_T9002


References

<biblio>

  1. 1 pmid=4913914
  2. 2 pmid=16224117
  3. 3 pmid=16076456
  4. 4 pmid=14559971
  5. 5 pmid=15183761

</biblio>