Team:Ruperto Carola/Contribution

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. [Martinez-Arias] [Davidow]. 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.

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.