Team:Bielefeld-CeBiTec/Demonstrate
Demonstrate your Work
The repurposing of a codon for the incorporation of a non-canonical amino acid (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/aminoacyl-tRNA synthetase (tRNA/aaRS) pair is necessary, which can charge the ncAA to the tRNA. We designed and synthetized the novel ncAA Nγ‑2‑cyanobenzothiazol‑6‑yl‑L‑asparagine (CBT-asparagine). This ncAA has the chemical ability of perform a highly specific covalent binding reaction, which we wanted to incorporate it into our target protein. Therefore, we created a library of aaRS with random mutagenized amino acid binding sites and a selection system to select for the aaRS that speciffally incorporates the ncAA. Next to the selection based approach, we modeled the aaRS which could incorporate our new amino acid CBT-asparagine. We showed that both ways are suitable for the evolution of aaRS.
Although incorporation of ncAAs through the amber codon works, there are a lot of problems with this approach. 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 use 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 was 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. We cloned a nucleoside triphosphate transporter that enables the uptake of both bases from the media. Furthermore, by transcriptome sequencing of the plant Croton tiglium, that produces isoG (also called crotonoside) as a defense mechanism, we found a suitable enzyme for the biosynthesis of isoG in E. coli. To detect the unnatural base we developed two orthogonal systems. A restriction experiment based on the software tool M.A.X. and an adaption of the Oxford Nanopore sequencing, which make up one software suite.
To demonstrate the possibilities offered by the incorporation of ncAAs, we developed a toolbox containing five different tools. We chose seven different ncAAs for these five tools and demonstrated interesting applications for them. These ncAAs can be used for various approaches in basic research, medicine and manufacturing. Furthermore, with our submitted parts, every iGEM team can incorporate these ncAAs into their target proteins.
Regarding our project, two of the ncAAs that are part of our toolbox perform an autocatalytic reaction upon irradiation with ultraviolet light. Therefore, we decided to build our own LED panel that allows us to perform experiments with these non‑canonical amino acids under reproducible irradiation conditions.
Achievements
Establishment of two orthogonal methods for the detection of unnatural base pairs in a target sequence via Oxford Nanopore sequencing and an enzyme based detection method
Development of a software suite for these orthogonal methods
Integration and characterization of the nucleotide transporter PtNTT2 from P.tricornutum in E.coli for the uptake of unnatural nucleoside triphosphates
Proof that certain Taq-polymerases can efficiently incorporate unnatural nucleotides
Construction of a toolbox consisting of five aminoacyl-tRNA synthetases for incorporation of non-canonical amino acids
Colocalization of the RuBisCo using a fluorescent amino acid
Development of a photoswitchable lycopene pathway
Design, chemical synthesis and proof of functionality of a novel, fully synthetic amino acid based on cyanonitrobenzothiazol and asparagine
Modeling more than ten new aaRS sequences
Library development with several hundred thousand sequences for selecting aminoacyl-tRNA synthetases
Construction of positive and negative selection plasmids for the evolution of new synthetases for non-canonical amino acids
Improvement of an aminoacyl-tRNA synthetase test-system by introducing a FRET-system and development of a ranking system
Construction of an LED panel for irradiating 96-well microtiter plates, which can be used to manipulate non-canonical amino acids and much more
Development of an Android App to control the LED panel with your smartphone via Bluetooth
Writing a biosafety report titled “Auxotrophy to Xeno-DNA: A Comprehensive Exploration of Combinatorial Mechanisms for a High-Fidelity Biosafety System”
Writing the ChImp Report on “Chances and Implications of an Expanded Genetic Code”
