The expansion of the genetic code is not solely confined to the incorporation of non-canonical amino acids into peptides, it also means the incorporation of unnatural base pairs (UBPs) into the DNA. The introduction of UBPs into the DNA results in a semisynthetic organism which has been a long-desired achievement in the field of synthetic biology. The research on UBPs bore several unnatural bases with different chemical properties. For our project we decided to use isoguanine (isoG) and 5-methyl-isocytosine (isoCm) to expand the genetic code. The establishment of novel protocols for standard lab methods like PCR and transformation were important in order to work with UBPs. Also, detection methods of UBPs in the DNA were required. 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 is available. Furthermore, we developed a two-plasmid-system for the 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. Within the meaning of synthetic biology, we also made a step towards an autonomous semisynthetic organism. Therefore, we conducted a transcriptome-based investigation to identify potential enzyme candidates responsible for the biosynthesis of isoG, which is part of the plant purine metabolism of Croton tiglium.
