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− | <p class="text-justify">
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− | Before proceeding with integrating our gene of interest we first characterized all the reported
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− | functions of the integration cassette. Therefore, the cassette was transformed into E.coli and grown on an ampicillin LB agar plate.
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− | Then we moved to portray the plasmid behavior in yeast. For that, the F102 strain containing 3
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− | auxotrophies (-leu2, -his2, and -ura3) was supplemented with the
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− | OrthoRep cassette and grown in different conditions. The leu2 gene dependency was first characcterized, as seen in the graphs below.
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− | </p>
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− | <h1>CAROUSEL</h1>
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− | Next, we moved to study the most tunable selection marker of the casette - the ura3 gene. To that effect, we generated a library of ura3 mutants using the selective pressure of 5-FOA. Here, the colonies were kept in the delta Leu medium for minimizing the selective pressure on ura3, thus
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− | forcing them to mutate the gene instead of losing the plasmid. After several weeks the colonies were transferred onto 5-FOA
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− | containing media to see if growth still occureds, which would indicate the successful mutation of the
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− | gene. The results of the experiment can be seen in our section <a href="https://2019.igem.org/Team:Ruperto_Carola/Results">Results</a>
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− | </p>
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− | As one can see, the complete inactivation of ura3 even without selective pressure took multiple
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− | weeks to obtain. But once achieved it remained relatively stable. Possible effects contributing to this
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− | result might be first the inhibition of nucleotide synthesis by 5-FOA, which "conserves mutations", as
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− | well as the large threshold due to multiple plasmids being present in the cell at the same time. The
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− | more complete explanation with the corresponding calculations can be obtained from our model. The observed effects proves ura3 to be an optimal selection marker for more complex evolving
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− | systems, which need an internal, easy to measure control for the mutation rate (which can be
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− | calculated using our model).</p>
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− | </div>
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− | <img class="img-fluid mx-auto d-block" src="https://static.igem.org/mediawiki/2019/2/2b/T--Ruperto_Carola--img-dice3.png" alt="">
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− | </div>
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− | <h2>characterization: polymerase BBa_K3173004</h2>
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− | <p class="text-justify">
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− | Leu2 is a gene coding for the enzyme 3-isopropylmalate dehydrogenase (also known as leu3) that on
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− | its turn is involved in the synthesis pathway of L-leucine. The gene presented here originates in yeast
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− | (S. cerevisiae), however, its close homologs can be found in several other organisms like the fungus
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− | Yarrowia lipolytica. [<x-ref>Martinez-Arias</x-ref>] [<x-ref>Davidow</x-ref>].
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− | In biotechnology, this gene is mostly used as an auxotrophy selection marker for different yeast
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− | strains. Therefore the gene is inserted into a plasmid and into an
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− | auxotrophic yeast strain and selected for in medium without leucine (delta leu medium). For
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− | providing the iGEM community more information on the selection markers we used for our project
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− | our team we characterized the leu2 gene in different media.
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− | In our case, the leu2 gene was inserted into the F102 strain lacking his2, leu2, and ura3 genes via an
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− | integration cassette as part of the OrthoRep system. The entire cassette can be found below in
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− | the OrthoRep section. The leu2, in this case, is a part of the plasmid section which is constantly
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− | mutagenized by the orthogonal polymerase TP DNAP1.
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− | For characterization, the strain transformed with the integration cassette together with other parts
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− | of the OrthoRep system was grown in delta uracil synthetic dropout medium with different
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− | concentrations of leucine ranging from 0 mg/l to 100 mg/l. For the growth assessment, the OD at
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− | 600nm was measured every hour in a plate reader. The results are presented in the following figure.
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− | As one can see the wildtype strain is performing much better in all the media. The F102 control strain
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− | without the plasmid does not grow at all, whereas the addition of the plasmid induces growth.
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− | However, the growth of the supplemented mutagenized strain is limited compared to the wildtype.
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− | This can be linked to two known phenomena: First, the high mutagenesis rate of the leu2 containing
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− | plasmids – which is expected from the experimental setup. Second, the poor ability of auxotrophic
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− | strains to take up missing nucleotides and amino acids from the supplied medium due to altered
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− | transporter pathways, which has been reported by several groups. In the
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− | case of the studied F102 strain, these are the missing uracil and histidine which are supplemented on
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− | the synthetic media.
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− | Therefore, for optimizing experimental conditions one has to account for such growth changes in
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− | auxotrophic strains and optimize experimental conditions accordingly. This can be done by either
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− | minimizing the number of auxotrophies in the employed strain via transformation of the
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− | corresponding genes or switching to a different strain. Nevertheless, the presented experiment
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− | proves that even constantly altered this part does perform its initial function, namely substituting the
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− | missing genomic leu2 gene and providing for the growth of the auxotrophic strain in the delta leu
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− | medium. Therefore, this part can be used as a reliable marker in yeast growth assays involving
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− | complex mutagenesis screening setups in high copy plasmids such as gene library generation via
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− | OrthoRep.</p>
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− | </div>
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− | </div>
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− | </div>
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− | </div>
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− | </div>
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