Our control for this lab was the EBY100 yeast strain of S. cerevisiae. Cultures of EBY100 (some transformed with GFP) were grown in liquid cultures of S Raffinose media and S Raffinose/1% Galactose overnight and examined the next day on the microscope. From our observations under the microscope, EBY100 rarely aggregate into clusters, while occasionally forming clumps of several cells.
Cadherin
Since cadherins only form dimers under the presence of calcium ions, we were able to use that variable as a second control mechanism. We observed that EBY100 transformed with cadherin would not form clumps until calcium was added to the cultures. However, when EDTA was added to the cultures, the clusters seen previously had dispersed.
HWP1
We did not have a clear understanding of how HWP1 really bind in Candida albicans and help form biofilms. So at first we just simply grew HWP1 in liquid cultures and we saw enormous clusters. However, we were unsure if the clumping was really caused by random adhesion between HWP1 or just adhesion to the budding from an EBY100 cell expressing HWP1. We then mixed EBY100 expressing HWP1 and EBY100 expressing GFP and HWP1 to see if the clusters were actually formed by cells randomly adhering to one and other. And from our observations, we were able to conclude that the clumping was indeed random. We also mixed normal EBY100 with EBY100 transformed with HWP1 and GFP to test if HWP1 was adhering just to the surface of EBY100, but we haven’t tested this hypothesis enough to reach a decisive conclusion.
Mgfp5
Since Mgfp5 is an adhesive protein from mussel, we believed that if we added more salt to the liquid cultures, the cells expressing the proteins would have a higher chance of adhering. We discovered that an increase of salt in the liquid cultures caused larger clumps.
Other Proteins
We also tried to express many other proteins but they did not function the way we expected them to when we observed the cells under the microscope. Eap1 was supposed to bind itself, but it didn’t. We thought that ZipRR and ZipEE, both leucine zippers, would to bind to one another, but they didn’t. ALS3 was believed to bind to itself and mammalian proteins, but it wasn’t show to bind to itself under the microscope. Cul3 is a hydrophobic protein but we were unable to determine if the cells expressing Cul3 were floating in our microscopy tests. BCL2 and BIM were supposed to adhere to each other but they were unsuccessful from what we observed under the microscope.
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