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Revision as of 16:39, 1 November 2017
During our project we worked on many different scientific fields, to find suitable ways to incorporate non-canonical amino acids into proteins. Thus, we needed to repurpose one of the existing codons and incorporated two new bases to create new codons in E. coli and we realized both ways.
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), with the chemical ability of fulfil a highly specific covalent binding reaction, and wanted to incorporate it into our target protein. Thus, and for the evolution of aaRS for the incorporation of any novel ncAA, we created a library of aaRS with random mutagenized amino acid binding sites and a selection system to select for the aaRS that could incorporate the ncAA specific. Next to the selection, 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.
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 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, we found a suitable enzyme for the biosynthesis of isoG in E. coli by a RNA sequencing of the plant Croton tiglium, that produces isoG. 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 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.
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), with the chemical ability of fulfil a highly specific covalent binding reaction, and wanted to incorporate it into our target protein. Thus, and for the evolution of aaRS for the incorporation of any novel ncAA, we created a library of aaRS with random mutagenized amino acid binding sites and a selection system to select for the aaRS that could incorporate the ncAA specific. Next to the selection, 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.
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 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, we found a suitable enzyme for the biosynthesis of isoG in E. coli by a RNA sequencing of the plant Croton tiglium, that produces isoG. 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 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.
