Assessing the biocompatibility of click-linked DNA in Escherichia coli.
The biocompatibility of a triazole mimic of the DNA phosphodiester linkage in Escherichia coli has been evaluated. The requirement for selective pressure on the click-containing gene was probed via a plasmid containing click DNA backbone linkages in each strand of the gene encoding the fluorescent protein mCherry. The effect of proximity of the click linkers on their biocompatibility was also probed by placing two click DNA linkers 4-bp apart at the region encoding the fluorophore of the fluorescent protein. The resulting click-containing plasmid was found to encode mCherry in E. coli at a similar level to the canonical equivalent. The ability of the cellular machinery to read through click-linked DNA was further probed by using the above click-linked plasmid to express mCherry using an in vitro transcription/translation system, and found to also be similar to that from canonical DNA. The yield and fluorescence of recombinant mCherry expressed from the click-linked plasmid was also compared to that from the canonical equivalent, and found to be the same. The biocompatibility of click DNA ligation sites at close proximity in a non-essential gene demonstrated in E. coli suggests the possibility of using click DNA ligation for the enzyme-free assembly of chemically modified genes and genomes.
Chemistry, University of Southampton, Southampton, SO17 1BJ, UK.
A Pia
AP
Sanzone
Afaf H
AH
El-Sagheer
Tom
T
Brown
Ali
A
Tavassoli
D057930:Click Chemistry; D004247:DNA / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics*; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000235:genetics; D014230:Triazoles / Q000737:chemistry; D000097573:Red Fluorescent Protein
D004247:DNA; D008164:Luminescent Proteins; D014230:Triazoles; D000097573:Red Fluorescent Protein