Results
E. coli Experiments
Goal
The goal of the E. coli experiments was to create our constructs and test their functionality before implementing them in our chosen target organism, S. cerevisiae.
Results
This phase of our project has primarily taught us the critical importance of media preparations for transformations. It is worth mentioning that, without this error, we would not have had confirmation that our backbone was functioning as intended. We also came in contact with the issues encountered setting up a proper colony PCR. At first we tried amplifying fragments larger than 3 kb, but we would recommend any team needing to perform colony PCRs to utilize fragments of sizes smaller than 1 kb as otherwise degradation or unspecific amplicons appear to be a problem. We would further advise any team using this backbone in the future to perform additional transformation frequency tests on non selective media as well as testing transformation efficiency for inserts of different length and on if the transformation efficiency of selective media might be dependent on the marker used. Regarding our backbone: If we would have had the time to further improve our backbone, we would have liked to introduce blunt cutters at the edges of our HO' homologies to have a more stable way of storing them and have an easier way of yielding higher concentrations that can be used for transformations. Our main takeaways from the second cycle are that specific primer design and the avoidance of multiple PCR products play a major role in cloning preparations and can, if not performed properly, prolong the time needed immensely. Also, we had issues in our cloning process as there was the possibility that the products we originally designed for S. cerevisiae expression would be producing toxic byproducts when expressed in E. coli. Due to time constraints we were not able to confirm if this is the case.
S. cerevisiae Experiments
Goal
During the S. cerevisiae experiments, we had to implement the constructs of the cloning experiments. In order to do that, we used the lithium method for the transformation1. The yeast strain BY4741 was used, along with selection mediums without leucine and uracil.
Results
Before starting the actual transformation of our construct, we conducted several transformations for practicing purposes. The medium we used for that was selective for leucine, because the training plasmid contained a corresponding insert. Unfortunately, this selection medium ended up having a lower concentration of agarose than actually needed. Because of that, we had troubles while plating and the results were difficult to see clearly. The control plates have not shown any colonies at all, which means that our selection medium performed in the anticipated way.
The first actual transfer with our construct showed a larger number of yeast colonies on each selective plate than expected. The high number of colonies suggests that the dropout medium unintentionally contained uracil.
The subsequent transformation took place on a medium that was produced using only new products and demonstrably contained no uracil. This resulted in no colonies being visible on the plates with each transformation. Our backbone normally should have an insert for uracil. That is why the result of the second transformation suggests that the previously cloned backbone did not contain an insert for uracil.
Finally, 28 colonies from the first transition were subjected to colony PCR. This was also done with 28 colonies of the yeast strain BY4741 in order to obtain a reference. The subsequent gel electrophoresis showed that only 4 of the 28 colonies did not have this insert implemented. The resulting evaluation showed a transformation frequency of approximately 85.7% for the sample used.
Plant Experiments
Goal
During our plant experiments we had to observe and find correlation with growth of three main variables: copper concentration influence, phosphate concentration influence and yeast influence on plant growth.
Results
After analyzing the data collected during the growth phase of C. pepo plants, we came to the following conclusions. Firstly, two different copper concentrations were tested: 0.5 μM, which is the normal level of copper in soil, and 100 μM. We tested the effect of different copper concentrations because it is an element we can use to trigger the mechanism of our modified organism. Based on statistical analysis of both height and biomass of C. pepo plants, the effect of copper on total growth (defined by these two factors) is not statistically significant and we could use copper as an activator for the system without any effect on plant growth. Secondly, we tested the effect of phosphate on growth, documenting plant height, biomass and leaf number, for which most of our test results were not statistically significant, which is not consistent with the literature. Finally, we analyzed whether S. cerevisiae present in the soil can influence plant growth based on plant height, biomass and leaf number. There was no statistically significant difference in plant height or number of leaves formed. However, there was one pair of groups compared on biomass that showed a statistical significant difference between plants grown with and without S. cerevisiae at the same soil phosphate concentration [0.5 mM], which is also in line with the literature. However, the majority of the groups in this comparison did not show a statistically significant difference.
We also measured the phosphate content of the plants using a Malachite Green Assay to understand the difference between phosphate concentrations in soils with and without S. cerevisiae. We observed a tendency for more phosphate to be detected in the run-off water in the pots that also contained yeast. However, the amount and accuracy of our data is low.
- Hegemann JH, Heick SB, Pöhlmann J, Langen MM, Fleig U. Targeted gene deletion in Saccharomyces cerevisiae and Schizosaccharomyces pombe. Methods Mol Biol. 2014;1163:45-73. doi: 10.1007/978-1-4939-0799-1_5. PMID: 24841299.↩