Difference between revisions of "Team:Bielefeld-CeBiTec/Project/Overview"

At the moment, protein design in limited to 20 to 22 canonical amino acids. The chemical abilities of these canonical amino acids are limited, thus a lot of interesting functions are not feasible in protein design. We want to change that and enable the incorporation of non-canonical amino acids with a broad range of additional abilities. There is only one problem: The DNA encodes the proteins with the four bases adenine, thymine, cytosine and guanosine in base triplets. All combinations of these triplets already code for a canonical amino acid or a translational stop. Thus, we need to repurpose one of the existing codons or incorporate a new base to create new codons in E. coli.

To solve this problem we want to try two different ways. The repurposing of a codon for the incorporation of a non-canonical amino (ncAA) is possible using the rarely used amber stop codon UAG or other rarely used codons like the leucine codon CUA. To incorporate a non-canonical amino acid using these codons, an orthogonal tRNA-aminacyl synthetase (aaRS) is necessary, which can charge the ncAA to the tRNA. We want to design a a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Project/translational_system/library_and_selection">library of aaRS and a suitable selection system to select the aaRS which incorporate the amino acid we want to use. To demonstrate this tool, we want to synthetize a novel amino acid which could build a specific covalent bond to another ncAA. Another attempt to find the matching aaRS for this ncAA will be a modeling of the aaRS structure.

Despite the incorporation of ncAAs through the amber codon works, there are a lot of problems. The repurposing of codons leads to the decrease of the growth of E. coli and it is only possible to incorporate two ncAAs. We decided to try a new way to incorporate ncAAs, the incorporation of an unnatural base pair into the DNA that encodes for 64 new codons. Our first challenge will be the uptake of the unnatural base from the media, because E.coli has no nucleoside triphosphate transporter and is not able to synthetize the bases itself. Our aim is to find a suitable transporter for the uptake or a biosynthesis pathway. A promising native host for the biosynthesis pathway is the plant Croton tiglium, that produces isoG. We want to sequence the C. tiglium transcript and test different enzyme variants which could catalyse the reaction to the unnatural base. Furthermore, we want to develop a detection system for the detection of the unnatural base.

To demonstrate the possibilities offered by the incorporation of ncAAs want to develop a toolbox containing different aaRS to incorporate different ncAAs and demonstrated interesting applications for them. These ncAAs are suitable for various approaches in basic research, medicine and manufacturing. Furthermore, with our submitted parts, every iGEM team could incorporate these ncAAs into their target proteins and expand the possibilities of their protein design.