Part:BBa_M36124:Experience

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Applications of BBa_M36124

Part BBa_M36124 was created with the goal of manipulating the glycolysis pathway in yeast. The part was designed after the native glucose-6-phosphate isomerase (phosphoglucose isomerase, or PGI) gene, which codes for the PGI enzyme that catalyzes the second step in glycolysis, the isomerization of glucose-6-phosphate to fructose-6-phosphate. We thought that the overexpression of PGI would cause the isomerization reaction to proceed at a faster rate, potentially opening up an anabolic pathway for fructose-6-phosphate to plain fructose, which is known to be the sweetest of the natural sugars. We also considered that an increase in isomerization rate due to an increase in PGI would help push glycolysis along faster if the yeast were growing in high-sugar media, up to growth-inhibitory concentrations of around 10% glucose. To test these hypotheses we put part BBa_M36124 into two different vectors with constitutive promoters of different strength. Both of these vectors contained identical Leucine and kanamycin selection, as well as ribosome binding site and termination site. For a control we used a similar vector, also with the same leucine and kanamycin selection, that had a GFP coding region following a rabinose-activated and glucose-repressible promoter, so we could be fairly certain that the vector would not affect the metabolism of yeast any more than the background for our experimental vectors. The strong-promoter vector was pD1211, which had a TEF1 promoter. The weak-promoter vector was pD1221, which had an ADH promoter. Both were made available from synthesis by DNA2.0. Our experimental assays consisted of an OD600 cell density assay combined with a fructose-6-phosphate fluorescence assay from BioVision. Duplicates of each of the three yeast lines containing one of each of our plasmids were grown for one day and two days in synthetic leucine drop-out media with 0.001%, 0.01%, 0.1%, 1%, 3.25%, 5.5%, 7.75%, and 10% glucose concentrations in separate inoculations. Results of both assays were inconclusive, especially for fluorescence levels corrected by cell density. Data is shown below.

Our results showed that our experimental design and execution need improvement to show any reliable data. This could lie in developing a high throughput culture technique to improve efficiency and keep culture aeration conditions consistent, finding a better fructose-6-phosphate fluorescence assay, and doing a total carbohydrate analysis with the phenol-sulfuric acid method. These things should be kept in mind in any future work with our standard biological part.

Note: The transformation efficiency of our vectors into S. cerevisiae improved significantly with the addition of 5% DMSO to the heat-shock solution. Groups wanting to transform one of those vectors with PGI should refer to a yeast transformation protocol that calls for 1-5% DMSO.

User Reviews

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