Conclusion

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Through our experiments, and further research, our original hypothesis was proven false. The growth rate of S. cerevisiae is not severely stunted by a single knockout of the gene SDH4. However, with further research, Kubo et al. conducted research showing that a double knockout of SDH4 (knocking out SDH1-4 and the sub gene SDH4b) deactivates the original gene as well as the sub gene that becomes activated in the absence of SDH4. This double knockout showed severely stunted growth in Kubo’s et al. research.

In regards to the novel analytical platforms, the petri dish-pipette tip model showed the most success and signs of growth. Thicker barriers were also more helpful in creating barriers and wax paper was incredibly cost efficient, as well as easily accessible; PDMS molds were shown to be cost efficient as well, as we used the same mold for both trials. The molds are easily washed out once an experiment is completed.

Our group would like to thank Dr. Van Dyken and Dr. Knecht for the instruction they have provided for us during this semester and guiding us. We would also like to thank the Howard Hughes Medical Institute for providing the program and opportunity of conduct this research. Last but not least, we would also like to thank Lily Acheapomaa-Piasare,  Yi Wen and Reid Collis for assisting and guiding us in all our research endeavors.

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PDMS Mold Trials: Success!

On our second trial of attempting growth on the PDMS mold, growth was seen!

To prevent the drying out that was experienced earlier, deionized water was pipetted directly into the surrounding areas of the mold into the petri dish. Since the mold was a few millimeters tall, the channels were not in contact with the DI water. This was successful in keeping moisture within the plate and in the channels. After two days, colonies of wild type yeast were observed within the channels, although they did not grow quite as linearly as expected. However, this was still a successful assay in producing growth and qualitative measurements. Further improvements upon this design may include increasing the size of the mold overall.

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Paper Assay Comparing WT and KO Strains

Our updated wax paper assay contained wax, hydrophobic barriers on all areas where yeast was not being plated, increasing the security of the barriers so the solid YPD would not spill off the assay. The wax paper assay was glued to the pipette tips and 1000  μl of solid YPD was pipetted onto all six squares, as well as 150  μl of wild type in three squares and 150  μl of the knockout strain in the other three squares. Growth was measured over 2 days. Growth for both types was observed, however there was a strange smell coming from within the petri dish, causing us to suspect possible contamination in this assay.

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Growth in Paper Assays on 11/14

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After four days of growth, the assay showed signs of possible yeast growth under a UV light. This completed our optimization, and assays with thicker wax barriers over the petri dish pipette tip model were printed to compare growth between the wild type strain and the knockout strain of S. cerevisiae.

The graph below displays the difference in growth rate over the four days.

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Mutant vs Wild Type Yeast Strain Experiment

Now that we found that our newest model design will promote growth, we are using the design to test growth in our knockout strain versus wild type strain.  This time, on the paper, we added a grid with boxes .5cm x .5cm to be able to measure our growth by area.  We will check the progress of our growth in two days, on Friday, and then in our next lab on Monday (November 14).

 

In the knockout strain row, there are 400 microliters of agar (found to be most successful in our last experiment) and 150 microliters of yeast.  In the wild type row, there are also 400 microliters of agar.  To match the number of cells, we pipetted 158 microliters of wild type yeast to make up for the difference in optical density (calculated using 1.02 x 150 microliters = .97 x ___ microliters).

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Success!

For the first time ever, we have actual results to report (our excitement cannot be overstated).

As show below, our yeast on paper assays grew! It is shown here, under normal lighting and under UV:

 

As for our PDMS mold, the agar dried out:

 

Improved Design

We continually experiences problems with our paper assay drying out and leaking outside of the hydrophobic barriers.  To fix this problem, we created a new design.  We cut pipette tips to create 5 stands within this dish to hold up the paper assay about 1 cm.  We then poured water on the bottom of the dish freely to provide a water source.  The wax printed circles are now bigger and contain varying amounts of YPD media (300, 350, and 400 microliters, with triplicates).  150 microliters of yeast were added to each circle.  We are incubating the dish at 30 degrees Celsius for 2 days.