Difference between revisions of "Team:Bielefeld-CeBiTec/Project/unnatural base pair"
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| + | <h2>Unnatural Base Pair Overview</h2> | ||
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The expansion of the genetic code is not solely confined to the incorporation of | The expansion of the genetic code is not solely confined to the incorporation of | ||
| − | <a target=„_blank“ href= | + | <a target=„_blank“ href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Project/translational_system">non-canonical amino acids</a> into peptides, |
| − | it also means the incorporation of <a target=„_blank“ href= | + | it also means the incorporation of <a target=„_blank“ href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Project/unnatural_base_pair/unnatural_base_pairs"> |
unnatural base pairs (UBPs)</a> into the DNA. The introduction of UBPs into the DNA results in a semisynthetic organism which has been a long-desired | unnatural base pairs (UBPs)</a> into the DNA. The introduction of UBPs into the DNA results in a semisynthetic organism which has been a long-desired | ||
achievement in the field of synthetic biology. The research on UBPs bore several unnatural bases with different chemical properties. | achievement in the field of synthetic biology. The research on UBPs bore several unnatural bases with different chemical properties. | ||
For our project we decided to use isoguanine (isoG) and 5-methyl-isocytosine (isoC<sup>m</sup>) to expand the genetic code. | For our project we decided to use isoguanine (isoG) and 5-methyl-isocytosine (isoC<sup>m</sup>) to expand the genetic code. | ||
| − | The establishment of <a target=„_blank“ href= | + | The establishment of <a target=„_blank“ href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Project/unnatural_base_pair/challenges">novel |
| − | protocols</a> for <a target=„_blank“ href= | + | protocols</a> for <a target=„_blank“ href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Notebook/Methods">standard lab methods</a> like |
PCR and transformation were important in order to work with UBPs. Also, detection methods of UBPs in the DNA were required. | PCR and transformation were important in order to work with UBPs. Also, detection methods of UBPs in the DNA were required. | ||
<br> | <br> | ||
| − | We were able to establish <a target=„_blank“ href= | + | We were able to establish <a target=„_blank“ href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Software">Oxford Nanopore Sequencing</a> for |
isoG and isoC<sup>m</sup> and developed a software to handle the data of unnatural bases. | isoG and isoC<sup>m</sup> and developed a software to handle the data of unnatural bases. | ||
| − | We also developed the software <a target=„_blank“ href= | + | We also developed the software <a target=„_blank“ href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Software#MAX">M.A.X.</a> that |
suggests a set of restriction enzymes based on the neighboring DNA sequence of the UBP. With these restriction enzymes, | suggests a set of restriction enzymes based on the neighboring DNA sequence of the UBP. With these restriction enzymes, | ||
a simple approach for retention or loss of the UBP <i>in vitro</i> is available. Furthermore, | a simple approach for retention or loss of the UBP <i>in vitro</i> is available. Furthermore, | ||
| − | we developed a <a target=„_blank“ href= | + | we developed a <a target=„_blank“ href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Results/unnatural_base_pair/preservation_system">two-plasmid-system</a> |
| − | for <a target=„_blank“ href= | + | for <a target=„_blank“ href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Project/unnatural_base_pair/preservation_system">UBP retention</a> <i>in vivo</i>. |
These plasmids encode for the nucleotide transporter | These plasmids encode for the nucleotide transporter | ||
| − | <a target=„_blank“ href= | + | <a target=„_blank“ href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Results/unnatural_base_pair/uptake"><i>Pt</i>NTT2</a>, |
Cas9 and a set of sgRNAs. <i>Pt</i>NTT2, taken from the algae <i>Phaeodactylum tricornutum</i>, | Cas9 and a set of sgRNAs. <i>Pt</i>NTT2, taken from the algae <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 | ensures the uptake of the unnatural triphosphates from the media. Plasmids that lose the UBP get cut by Cas9, because the sgRNAs bind to | ||
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We also made a step towards an autonomous semisynthetic organism. | We also made a step towards an autonomous semisynthetic organism. | ||
From literature, we identified a natural source of the "unnatural" base isoG, the plant | From literature, we identified a natural source of the "unnatural" base isoG, the plant | ||
| − | <a target=„_blank“ href= | + | <a target=„_blank“ href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Results/unnatural_base_pair/biosynthesis"><i>Croton tiglium</i></a>. |
We conducted a transcriptome-based investigation to identify potential enzyme candidates responsible for the biosynthesis of isoG, | We conducted a transcriptome-based investigation to identify potential enzyme candidates responsible for the biosynthesis of isoG, | ||
which we then heterologously expressed in <i>E. coli</i> and characterized <i>in vitro</i>. </div> | which we then heterologously expressed in <i>E. coli</i> and characterized <i>in vitro</i>. </div> | ||
Latest revision as of 02:56, 2 November 2017
Unnatural Base Pair Overview
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. With these restriction enzymes, a simple approach for retention or loss of the UBP in vitro is available. Furthermore, we developed a two-plasmid-system for UBP retention in vivo. These plasmids encode for the nucleotide transporter PtNTT2, Cas9 and a set of sgRNAs. PtNTT2, taken from the algae 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 in the preservation of the UBP in vivo. We also made a step towards an autonomous semisynthetic organism. From literature, we identified a natural source of the "unnatural" base isoG, the plant Croton tiglium. We conducted a transcriptome-based investigation to identify potential enzyme candidates responsible for the biosynthesis of isoG, which we then heterologously expressed in E. coli and characterized in vitro.
