Difference between revisions of "Team:Bielefeld-CeBiTec/Project/unnatural base pair"

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Unnatural Base Pairs
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Unnatural Base Pairs Overview
 
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          <h3> Overview </h3>
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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 into a semisynthetic organism which has been a long desired achievement in the field of synthetic biology. The research on UBPs beared several unnatural bases with different chemical properties. For our approach we decided to use isoguanine (isoG) and 5-methyl-isocytosine (isoC<sup>m</sup>) to expand the genetic code. The establishment of novel protocols for standard lab methods like PCR and transformation were necessary 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 isoC<sup>m</sup> 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. A very simple analysis for retention or loss of the UBP is now possible due to the help of these restriction enzymes. Furthermore, we developed a two-plasmid-system for the UBP retention in vivo. These plasmids code for the nucleotide transporter <i>Pt</i>NTT2, Cas9 and a set of sgRNAs. <i>Pt</i>NTT2, taken from the alga <i>Phaeodactylum tricornutum</i>, 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 into 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 investigated the biosynthesis pathway of isoG, which is part of the plant metabolism of <i>L. croton tiglium</i>.
 
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 into a semisynthetic organism which has been a long desired achievement in the field of synthetic biology. The research on UBPs beared several unnatural bases with different chemical properties. For our approach we decided to use isoguanine (isoG) and 5-methyl-isocytosine (isoC<sup>m</sup>) to expand the genetic code. The establishment of novel protocols for standard lab methods like PCR and transformation were necessary 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 isoC<sup>m</sup> 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. A very simple analysis for retention or loss of the UBP is now possible due to the help of these restriction enzymes. Furthermore, we developed a two-plasmid-system for the UBP retention in vivo. These plasmids code for the nucleotide transporter <i>Pt</i>NTT2, Cas9 and a set of sgRNAs. <i>Pt</i>NTT2, taken from the alga <i>Phaeodactylum tricornutum</i>, 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 into 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 investigated the biosynthesis pathway of isoG, which is part of the plant metabolism of <i>L. croton tiglium</i>.

Revision as of 17:05, 27 October 2017

Unnatural Base Pairs Overview
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 into a semisynthetic organism which has been a long desired achievement in the field of synthetic biology. The research on UBPs beared several unnatural bases with different chemical properties. For our approach 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 necessary 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. A very simple analysis for retention or loss of the UBP is now possible due to the help of these restriction enzymes. Furthermore, we developed a two-plasmid-system for the UBP retention in vivo. These plasmids code for the nucleotide transporter PtNTT2, Cas9 and a set of sgRNAs. PtNTT2, taken from the alga 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 into 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 investigated the biosynthesis pathway of isoG, which is part of the plant metabolism of L. croton tiglium.