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Team:Warsaw/SyntheticCloning
From 2011.igem.org
Synthetic cloning and phi29 amplification as a new safety standard
Generating large collection of biological parts requires processing large number of DNA constructs. Moreover one of the primary goals of the collections is sharing parts e.g. sending to researchers or to iGEM teams. To share parts is is obligatory to amplify DNA via transforming DNA to living cells. Some of the construct may be pathogenic. Our team last Some of the parts may be newly synthesized parts or natural yet not fully tested. Those parts are potentially hazardous. Moreover GMO bacteria can escape to the environment. We developed a safe techniques of cloning and handling large collection of DNA parts without use of any living cells making it safe to researchers and the environment.
Safe storing and sharing of biological parts
We used phi29 rolling circle amplification to amplify DNA from the distribution. It performs rolling circle amplification of the circular plasmids, has high fidelity and procesivity. We managed to get lots of DNA in 2 hours. Protocol and experimental results ****here****. It is safe to use with pathogenic ad toxic genes. We would recommend it to everyone who works with collection of parts that are potentially dangerous. Also to everyone who value time.
Safe Synthetic Cloning meets the needs of biologists
Molecular cloning techniques require propagation of the construct in living cells that is transforming plasmids into e.g. E. coli and growing cultures overnight.
- It is a time-consuming process and can't be easily sped up. We can speed up digestion by using fast enzymes and speed up gel electrophoresis by using lithium-borate buffer, but it is hard to make cells grow faster
- Genes toxic to the new host (e.g. E. coli) are difficult to clone this way. Actually over-expression of naturally occurring proteins e.g. membrane transporters can be evolutionary disadvantageous. It means that you are likely to get lots of empty vectors while cloning these proteins in E. coli
- Molecular clonig results in creation of genetically modified organisms at each cloning step. When working with potentially hazardous genes it is undesirable to have those genes transformed into cells without appropriate regulatory systems. It would be safer to construct genetically modified organisms only at the end of the process, when DNA constructs are ready. This is possible using DNA synthesis, but still reminds expensive.
Solution: phi29 polymerase based cell-free clonig
An interesting alternative is phi28 polymerase. It performs rolling circle amplification of the circular plasmids. It is processive and has high fidelity resulting in error rate about 10 to -7[1]. Although an error by phi29 DNA polymerase could occur, the error would not be exponentially amplified as in PCR.[2]
- This is why amplification using phi29 polymerase can be use instead of living cells in cell free cloning.[3]
- Moreover, phi29 amplification products can be used directly to synthesize protein in cell-free transcription-translation systems.[4]
- This opens up a possibility of wet lab experiments without creation of genetically modified organisms, when it is not necessary.
- Esteban J, Salas M, Blanco L. Fidelity of Phi29 DNA polymerase. J Biol Chem 1993; 268:2719–2726.
- Reagin MJ, Giesler TL, Merla AL, Resetar-Gerke JM, Kapolka KM, Mamone JA TempliPhi: A sequencing template preparation procedure that eliminates overnight cultures and DNA purification. J Biomol Tech. 2003 Jun.
- Takahashi H, Yamamoto K, Ohtani T, Sugiyama S. Cell-free cloning using multiply-primed rolling circle amplification with modified RNA primers. Biotechniques. 2009 Jul.
- Kumar G, Chernaya G. Cell-free protein synthesis using multiply-primed rolling circle amplification products. Biotechniques. 2009 Jul.
