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+ | <h1>Parts</h1> | ||
+ | </div> | ||
+ | <p> | ||
+ | We aimed to characterize the performance of the leucene gene present in the integration cassette introduced into our auxotrophic yeast strains. | ||
+ | </p> | ||
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+ | <h2>characterization: polymerase BBa_K3173004</h2> | ||
+ | <p class="text-justify"> | ||
+ | Leu2 is a gene coding for the enzyme 3-isopropylmalate dehydrogenase (also known as leu3) that on | ||
+ | its turn is involved in the synthesis pathway of L-leucine. The gene presented here originates in yeast | ||
+ | (S. cerevisiae), however, its close homologs can be found in several other organisms like the fungus | ||
+ | Yarrowia lipolytica. [<x-ref>Martinez-Arias</x-ref>] [<x-ref>Davidow</x-ref>]. | ||
+ | In biotechnology, this gene is mostly used as an auxotrophy selection marker for different yeast | ||
+ | strains. Therefore the gene is inserted into a plasmid and into an | ||
+ | auxotrophic yeast strain and selected for in medium without leucine (delta leu medium). For | ||
+ | providing the iGEM community more information on the selection markers we used for our project | ||
+ | our team we characterized the leu2 gene in different media. | ||
+ | In our case, the leu2 gene was inserted into the F102 strain lacking his2, leu2, and ura3 genes via an | ||
+ | integration cassette as part of the OrthoRep system. The entire cassette can be found below in | ||
+ | the OrthoRep section. The leu2, in this case, is a part of the plasmid section which is constantly | ||
+ | mutagenized by the orthogonal polymerase TP DNAP1. | ||
+ | For characterization, the strain transformed with the integration cassette together with other parts | ||
+ | of the OrthoRep system was grown in delta uracil synthetic dropout medium with different | ||
+ | concentrations of leucine ranging from 0 mg/l to 100 mg/l. For the growth assessment, the OD at | ||
+ | 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 | ||
+ | without the plasmid does not grow at all, whereas the addition of the plasmid induces growth. | ||
+ | However, the growth of the supplemented mutagenized strain is limited compared to the wildtype. | ||
+ | This can be linked to two known phenomena: First, the high mutagenesis rate of the leu2 containing | ||
+ | plasmids – which is expected from the experimental setup. Second, the poor ability of auxotrophic | ||
+ | strains to take up missing nucleotides and amino acids from the supplied medium due to altered | ||
+ | transporter pathways, which has been reported by several groups. In the | ||
+ | case of the studied F102 strain, these are the missing uracil and histidine which are supplemented on | ||
+ | the synthetic media. | ||
+ | Therefore, for optimizing experimental conditions one has to account for such growth changes in | ||
+ | auxotrophic strains and optimize experimental conditions accordingly. This can be done by either | ||
+ | minimizing the number of auxotrophies in the employed strain via transformation of the | ||
+ | corresponding genes or switching to a different strain. Nevertheless, the presented experiment | ||
+ | proves that even constantly altered this part does perform its initial function, namely substituting the | ||
+ | missing genomic leu2 gene and providing for the growth of the auxotrophic strain in the delta leu | ||
+ | medium. Therefore, this part can be used as a reliable marker in yeast growth assays involving | ||
+ | complex mutagenesis screening setups in high copy plasmids such as gene library generation via | ||
+ | OrthoRep.</p> | ||
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+ | <groupparts>iGEM19 Ruperto_Carola</groupparts> | ||
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Revision as of 02:07, 13 December 2019
Parts
We aimed to characterize the performance of the leucene gene present in the integration cassette introduced into our auxotrophic yeast strains.
![](https://static.igem.org/mediawiki/2019/2/2b/T--Ruperto_Carola--img-dice3.png)
characterization: polymerase BBa_K3173004
Leu2 is a gene coding for the enzyme 3-isopropylmalate dehydrogenase (also known as leu3) that on
its turn is involved in the synthesis pathway of L-leucine. The gene presented here originates in yeast
(S. cerevisiae), however, its close homologs can be found in several other organisms like the fungus
Yarrowia lipolytica. [