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

Revision as of 01:28, 2 November 2017

Unnatural Base Pair Overview

Uptake and Biosynthesis

For the uptake of supplemented unnatural bases we used the nucleotide transporter PtNTT2. This unspecific nucleotide transporter is originated from the alga Phaeodactylum tricornutum, where it is located in the plastid membrane. Additional signal peptides guide PtNTT2 to be integrated within the plasma membrane of E. coli. This ensures the uptake of isoGTP and isoC^mTP from the surrounding medium into the cells.
Instead of feeding the cells with unnatural bases by supplementing them to medium, the biosynthesis of unnatural bases would be a huge step towards a fully autonomous semi-synthetic organism. The Botanical Garden of Marburg University kindly provided us some cuttings of the plant Croton tiglium, which is known to produce isoG as one of the unnatural bases we used. After an RNA extraction and RNA-sequencing library preparation, we identified some potential enzyme candidates based on the transcriptome, which could be responsible for the production of isoG.

Retention System

We designed a two-plasmid system for the retention of our unnatural base pair (UBP). The first plasmid contains the nucleotide transporter PtNTT2, and cas9 whereas the second plasmid contains the UBP and five sgRNAs. PtNTT2 ensures the uptake of unnatural nucleotides from the medium. Cas9 is needed to cleave all plasmids that lost the UBP on the second plasmid. There are five possible sequences after a mutation of the UBP. The UBP could either be substituted by one of the four natural bases or completely deleted. There is a sgRNA for all five cases that binds the sequence recruiting Cas9 to cut the plasmid. Therefore, the selection marker on the plasmids that carry the UBP leads to its retention.

Development of New Methods

The use of UBPs requires new protocols for standard lab methods. We had to adapt the PCR, and the transformation of plasmids with UBPs. Another challenge was the validation of the presence of the UBP. Therefore we created the software tool M.A.X. that identifies a set of restriction enzymes based on the input DNA sequence. In a simple in vitro test the predicted restriction enzymes can then be used to digest the DNA and a following gel electrophorese will indicate the presence or absence of the UBP according to M.A.X.

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 <h3> <a href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Results/unnatural_base_pair/uptake_and_biosynthesis">Uptake and Biosynthesis</a> </h3>
 <article>
-For the uptake of the unnatural bases we used the nucleotide transporter <i>Pt</i>NTT2. This unspecific nucleotide transporter is originated from the alga <i>Phaeodactylum tricornutum</i>, where it is located in the plastid membrane. Additional signal peptides guide <i>Pt</i>NTT2 to be integrated into the plasma membrane of <i>E. coli</i>. This ensures the uptake of isoGTP and isoC<sup>m</sup>TP from the surrounding media inside the cells.
+For the uptake of supplemented unnatural bases we used the nucleotide transporter <i>Pt</i>NTT2. This unspecific nucleotide transporter is originated from the alga <i>Phaeodactylum tricornutum</i>, where it is located in the plastid membrane. Additional signal peptides guide <i>Pt</i>NTT2 to be integrated within the plasma membrane of <i>E. coli</i>. This ensures the uptake of isoGTP and isoC<sup>m</sup>TP from the surrounding medium into the cells.
 <br>
-Instead of feeding the cells with unnatural bases by supplementing the media, the biosynthesis of unnatural bases would be a step towards a fully autonomous semi-synthetic organism. The Botanical garden of Marburg University kindly provided us with the plant <i>L. croton tiglium</i>, which is known to produce isoG as one of the unnatural bases we used. After an RNA extraction and RNA-sequencing library preparation, we identified some potential enzyme candidates based on the transcriptome, which could be responsible for the production of isoG.
+Instead of feeding the cells with unnatural bases by supplementing them to medium, the biosynthesis of unnatural bases would be a huge step towards a fully autonomous semi-synthetic organism. The Botanical Garden of Marburg University kindly provided us  some cuttings of the plant <i>Croton tiglium</i>, which is known to produce isoG as one of the unnatural bases we used. After an RNA extraction and RNA-sequencing library preparation, we identified some potential enzyme candidates based on the transcriptome, which could be responsible for the production of isoG.
 </article>
 </div>
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 <h3> <a href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Results/unnatural_base_pair/preservation_system">Retention System</a> </h3>
 <article>
-We designed a two-plasmid system for the retention of our unnatural base pair (UBP). The first plasmid contains the nucleotide transporter <i>Pt</i>NTT2, and <i>cas9</i> whereas the second plasmid contains the UBP and five sgRNAs. <i>Pt</i>NTT2 ensures the uptake of unnatural nucleotides from the media. <i>cas9</i> is needed to cut all plasmids that loses the UBP on the second plasmid. There are five possible sequences after a mutation of the UBP. The UBP could be replaced by one of the four natural base pairs or just completely deleted. There is a sgRNA for all five cases that binds the sequence recruiting Cas9 to cut the plasmid. Therefore the selection pressure on plasmids that carry the UBP leads to its retention.
+We designed a two-plasmid system for the retention of our unnatural base pair (UBP). The first plasmid contains the nucleotide transporter <i>Pt</i>NTT2, and <i>cas9</i> whereas the second plasmid contains the UBP and five sgRNAs. <i>Pt</i>NTT2 ensures the uptake of unnatural nucleotides from the medium. <i>Cas9</i> is needed to cleave all plasmids that lost the UBP on the second plasmid. There are five possible sequences after a mutation of the UBP. The UBP could either be substituted by one of the four natural bases or completely deleted. There is a sgRNA for all five cases that binds the sequence recruiting Cas9 to cut the plasmid. Therefore, the selection marker on the plasmids that carry the UBP leads to its retention.
 </article>
 </div>
@@ Line 45: / Line 45: @@
 <h3><a href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Results/unnatural_base_pair/development_of_new_methods">Development of New Methods</a></h3>
 <article>
-The use of UBPs requires new protocols for standard lab methods. We had to adapt the PCR, and the transformation of plasmids with UBPs. Another challenge was the analysis of the presence of the UBP. Therefore we created the software tool M.A.X. that finds a set of restriction enzymes based on the DNA sequence. This allows the simple <i>in vitro</i> test for the presence or absence of the UBP by looking at the pattern of the bands on a agarose gel electrophoresis after digesting the DNA according to M.A.X..
+The use of UBPs requires new protocols for standard lab methods. We had to adapt the PCR, and the transformation of plasmids with UBPs. Another challenge was the validation of the presence of the UBP. Therefore we created the software tool M.A.X. that identifies a set of restriction enzymes based on the input DNA sequence. In a simple <i>in vitro</i> test the predicted restriction enzymes can then be used to digest the DNA and a following gel electrophorese will indicate the presence or absence of the UBP according to M.A.X.
 </article>
 </div>