Team:Bielefeld-CeBiTec/Results/unnatural base pair

Unnatural Base Pair Overview

Uptake and Biosynthesis

For the uptake of the unnatural bases we used the nucleotide transporter PtNTT2. This unspecific nucleotide transporter is originated from the alga Phaeodactylum tricornutum, where it is located in the plastid membrane. Additional signal peptides guide PtNTT2 to be integrated into the plasma membrane of E. coli. This ensures the uptake of isoGTP and isoCmTP from the surrounding media inside the cells.
Instead of feeding the cells with unnatural bases by supplementing the media, the biosynthesis of unnatural bases would be a step towards a fully autonomous semi-synthetic organism. The Botanical garden of Marburg University kindly provided us with the plant L. croton tiglium, which is known to produce isoG as one of the unnatural bases we used. After an RNA extraction and RNA-sequencing library preparation, we identified some potential enzyme candidates based on the transcriptome, which could be responsible for the production of isoG.

Retention System

We designed a two-plasmid system for the retention of our unnatural base pair (UBP). The first plasmid contains the nucleotide transporter PtNTT2, and cas9 whereas the second plasmid contains the UBP and five sgRNAs. PtNTT2 ensures the uptake of unnatural nucleotides from the media. cas9 is needed to cut all plasmids that loses the UBP on the second plasmid. There are five possible sequences after a mutation of the UBP. The UBP could be replaced by one of the four natural base pairs or just completely deleted. There is a sgRNA for all five cases that binds the sequence recruiting Cas9 to cut the plasmid. Therefore the selection pressure on plasmids that carry the UBP leads to its retention.

Development of New Methods

The use of UBPs requires new protocols for standard lab methods. We had to adapt the PCR, and the transformation of plasmids with UBPs. Another challenge was the analysis of the presence of the UBP. Therefore we created the software tool M.A.X. that finds a set of restriction enzymes based on the DNA sequence. This allows the simple in vitro test for the presence or absence of the UBP by looking at the pattern of the bands on a agarose gel electrophoresis after digesting the DNA according to M.A.X..