Unnatural Base Pair Overview
We were able to establish Oxford Nanopore Sequencing for isoG and isoCm and developed a software to handle the data of unnatural bases. We also developed the software M.A.X. that suggests a set of restriction enzymes based on the neighboring DNA sequence of the UBP. With these restriction enzymes, a simple approach for retention or loss of the UBP in vitro is available. Furthermore, we developed a two-plasmid-system for UBP retention in vivo. These plasmids encode for the nucleotide transporter PtNTT2, Cas9 and a set of sgRNAs. PtNTT2, taken from the algae Phaeodactylum tricornutum, ensures the uptake of the unnatural triphosphates from the media. Plasmids that lose the UBP get cut by Cas9, because the sgRNAs bind to every DNA sequences that carries a mutation instead of the UBP. This results in the preservation of the UBP in vivo. We also made a step towards an autonomous semisynthetic organism. From literature, we identified a natural source of the "unnatural" base isoG, the plant Croton tiglium. We conducted a transcriptome-based investigation to identify potential enzyme candidates responsible for the biosynthesis of isoG, which we then heterologously expressed in E. coli and characterized in vitro.