Imperial/Cell-Free/Whatis
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== What is Cell-Free? == | == What is Cell-Free? == | ||
- | Cell- | + | '''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 [http://partsregistry.org/Main_Page Registry of Standard Biological Parts]. |
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+ | [[Image:IC07_central_dogma.jpg|thumb|300px|left|The Central Dogma (Picture by Andy Verstraete, 1999)]] | ||
+ | '''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. <cite>1</cite> Not only do CFS house the molecular machinery necessary for transcription and translation, they are also optimized for these two processes. | ||
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+ | '''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, an ATP regenerating system involving either creatine phosphate and creatine kinase or phosphoenolpyruvate and pyruvate kinase is used to power and prolong the lifespan of the expression machinery <cite>2</cite>. | ||
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+ | '''A good analogy''' compared this transcription-translation machinery of the CFS to the hardware and the synthetic DNA to the software. <cite>3</cite> This gives an apt illustration of the ease of building genetically engineered machines using the cell-free approach. Simply by adding the DNA template to the cell extract and feeding solution, the CFS would be able to express the encoded genetic circuit. | ||
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+ | '''The PURE system''' has recently been developed as a reconstituted CFS for synthesizing proteins using recombinant elements <cite>4</cite>. This purely synthetic expression system enables even better quality control over the reaction conditions. | ||
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As a forward step to stricter quality control, as well as the specifications of our projects necessitating a cell-free environment, part of our contributions to iGEM involve the investigation and characterization of cell-free expression systems as a new chassis for the Registry. | As a forward step to stricter quality control, as well as the specifications of our projects necessitating a cell-free environment, part of our contributions to iGEM involve the investigation and characterization of cell-free expression systems as a new chassis for the Registry. | ||
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<center> On to next stage: | [https://2007.igem.org/Imperial/Cell-Free/Comparison Cell-Free vs. Cell >>]</center> | <center> On to next stage: | [https://2007.igem.org/Imperial/Cell-Free/Comparison Cell-Free vs. Cell >>]</center> | ||
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== References == | == References == |
Revision as of 19:37, 26 October 2007
What is Cell-Free?
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 [http://partsregistry.org/Main_Page Registry of Standard Biological Parts].
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. 1 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, an ATP regenerating system involving either creatine phosphate and creatine kinase or phosphoenolpyruvate and pyruvate kinase is used to power and prolong the lifespan of the expression machinery 2.
A good analogy compared this transcription-translation machinery of the CFS to the hardware and the synthetic DNA to the software. 3 This gives an apt illustration of the ease of building genetically engineered machines using the cell-free approach. Simply by adding the DNA template to the cell extract and feeding solution, the CFS would be able to express the encoded genetic circuit.
The PURE system has recently been developed as a reconstituted CFS for synthesizing proteins using recombinant elements 4. This purely synthetic expression system enables even better quality control over the reaction conditions.
As a forward step to stricter quality control, as well as the specifications of our projects necessitating a cell-free environment, part of our contributions to iGEM involve the investigation and characterization of cell-free expression systems as a new chassis for the Registry.
Types of Extracts
In vitro synthesis of proteins using cell-free extracts consists of two main processes - transcription of DNA into messenger RNA (mRNA) and translation of mRNA into polypeptides. Coupled transcription-translation systems usually combine a bacteriophage RNA polymerase and promoter (T7, T3, or SP6) with eukaryotic or prokaryotic extracts. In addition, the [http://www.nature.com/nbt/journal/v19/n8/full/nbt0801_732.html PURE] system is a reconstituted CFS for synthesizing proteins using recombinant elements (2). |
Comparison between different types of cell extracts
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Types of Compartmentalization
Previous research has been done to optimize cell extracts for in vitro protein synthesis. Their endogenous genetic content is removed so that exogenous DNAs or mRNAs can be expressed. Nuclease activity has been reduced and degradation of certain amino acids has been identified. ATP regenerating systems have also been added to improve the energy supply. Different strategies of compartmentalization have been explored to prolong the lifespan of CFS.
Batch-mode CFS
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Continuous-exchange CFS
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Vesicle-encapsulated CFS
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References
- Noireaux V, Bar-Ziv R, and Libchaber A. Principles of cell-free genetic circuit assembly. Proc Natl Acad Sci U S A 2003 Oct 28; 100(22) 12672-7. doi:10.1073/pnas.2135496100 pmid:14559971.
- Yoshihiro Shimizu, Yutetsu Kuruma, Bei-Wen Ying, So Umekage, Takuya Ueda (2006) Cell-free translation systems for protein engineering FEBS Journal 273 (18), 4133–4140. doi:10.1111/j.1742-4658.2006.05431.x
- 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 pmid:16224117.