Click chemistry has the potential to be employed for the assembly of large DNA fragments, by purely chemical methods. However to enable this, the bio-compatibility of the resulting click-linked DNA must be examined. Click DNA linkers were incorporated into a plasmid within the gene encoding for an ampicillin resistance marker. The plasmid was transformed into E. coli and resulting colonies found to survive on LB agar plates supplemented with ampicillin. This indicated that the click DNA linker was replicated and transcribed correctly by the cellular machinery of E. coli. The observed bio-compatibility was further probed by demonstrating the functionality of the click-linked DNA in nucleotide excision repair deficient cells line. The bio-compatibility of the click DNA linker was then investigated in a non-essential gene by constructing a click-linked variant of the gene encoding for the fluorescent mCherry protein. Experiments carried out using a plasmid containing two click DNA linkers in the region of the gene encoding for the mCherry fluorophore provided further evidence that the click DNA linker was functional in E. coli. Moreover, using a coupled in vitro transcription/translation system the yield and fluorescence of the mCherry protein expressed from the plasmid containing the click DNA linker was similar to that from canonical DNA. Investigation of the bio-compatibility of the click DNA linker in mammalian cells showed that a plasmid containing the click-linked DNA, had the same viability of a plasmid containing canonical DNA. Finally, the use of click ligation for the assembly of the first 229 bp of the mCherry gene was investigated. Two different approaches referred as “templated click assembly” and “one-pot click assembly” were employed and the bio-compatibility of the click DNA linker was confirmed in both cases.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:581539 |
| Date | January 2013 |
| Creators | Sanzone, A. Pia |
| Contributors | Tavassoli, Ali |
| Publisher | University of Southampton |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://eprints.soton.ac.uk/355714/ |
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