Team:Warsaw/SyntheticCloning

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.

A way to skip those problems would be to amplify the construct using PCR, but taq polymerase has error rate about 1 in 9,000 nucleotides. Since the early PCR products are also templates in the PCR reaction the errors accumulate.

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.

Want to perform cell-free cloning? See here for technical advice.

Getting biobrick from the distribution requires transformation and DNA isolation. We decided to save some time and to use phi29 polymerase based amplification to get a biobrick from the distribution suitable for cloning. We managed to get lots of DNA in 2 hours. Protocol and experimental results here.

1. Esteban J, Salas M, Blanco L. Fidelity of Phi29 DNA polymerase. J Biol Chem 1993; 268:2719–2726.
2. 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.
3. Takahashi H, Yamamoto K, Ohtani T, Sugiyama S. Cell-free cloning using multiply-primed rolling circle amplification with modified RNA primers. Biotechniques. 2009 Jul.
4. Kumar G, Chernaya G. Cell-free protein synthesis using multiply-primed rolling circle amplification products. Biotechniques. 2009 Jul.