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This update was very helpful in first understanding the basis of our research. It very simply explained the parts involved in auxin hormone signaling. It highlighted the very diverse applications of both using the naturally-occurring and manipulating the auxin-pathway. It was helpful to know that many of the targeted proteins for degradation are unknown and that it could be expanded to hormone signaling in humans. Because there are over 1,000 E3 ligases, there really are innumerable possible UPS complexes, giving us a lot of creativity and encouragement to experiment with virtually any protein of interest, as well as their receptors. Because the selective degradation of proteins via the auxin pathway is dependent on varying concentrations over time and alterations, research and use of protein degradation within these constraints would work nicely with the highly dynamic and variable environment of human metabolic pathways. Despite this broad variability, however, this pathway is very tightly regulated and can be controlled for various magnitudes and degradation, creating a very elastic tool for research. Because hormone signaling is never one input:one output, the possible cross-talk that would ensue should always be in the back of our minds to avoid any unwanted outcomes or to anticipate beneficial responses.

This research article is what we based our constructs off of. We made sure that we could at least design these same constructs to have a library for screening and analysis that we know would work before continuing with our novel experimentation. The article brought to light that “interfering directly at the protein level” would be a more reasonable approach to controlling protein levels in comparison to gene removal or downregulation. This is because, by using the auxin pathway, responses are reversible and quicker, responding in minutes as opposed to hours. Because the components of the auxin pathway are highly conserved sequences among eukaryotes, it should result in easy and efficient manipulation and control due to the advances in biochemistry. Also, this TIR1 (auxin receptor) component found naturally in plants is also capable of activating E3 in other organisms, which would allow for human or higher eukaryotic applications. For our research, we are looking to degrade myostatin to help those with muscular dystrophy and other diseases of hypotonia. Because these responses are in minutes, we also thought it could aid in the degradation of gluten, which is something people with Celiac cannot do. Because humans do not have auxin-dependent systems, introducing these constructs to degrade proteins of interest would not have adverse off-target effects. It was also mentioned that full-length and truncated versions of the AID-tags function efficiently and I believe that full-length or longer versions will be necessary for the larger yeast genomes we are interested in, whereas the truncated version will work fine with E. coli. Various methods of screening when antibodies are not available are also mentioned.

This article was a good reminder that although the first thought in regulating gene expression is to manipulate transcription, often the easier and less adverse method is post-translational modification. In this case, utilizing the UPS in plants and vertebrates provides a robust and dynamic method to regulate protein expression rates. This can be done by modification via ubiquitination, or by complete deletion via the 26s proteasome. This commentary brought to light that F-box E3 genes are rapidly evolving, both a daunting and exciting quality. E3s apparently are very conserved from yeast to humans, which will be a great support for our future goals of therapeutic applications in humans for various pathologies. One of the great benefits of using the auxin hormone pathway as a means of protein regulation is that varying concentrations of auxin, and the many substrates it can accommodate, give us a bounty of possible proteins for targeted degradation, while working with the varying environment of biological organisms. Because humans do not have the auxin pathway, this method of protein degradation will not have adverse effects in human subjects for it has nothing to activate or repress. This article mentions that dysregualtion of the human UPS is linked to some pathologies, but that little progress has been made through the E3s, further motivating us to make this construct work! I learned that auxin does not catalyze its receptor via conformational change but rather it increases the affinity for the receptor to the protein of interest by nucleating a hydrophobic core. This creates a co-receptor system that further illustrates the equally weighted importance of every component of this system; the ligase, the peptide, and the hormone are all required for degradation to occur. This article brought to light that auxin utilizes a co-factor, inositol hexakisphosphate (InsP6), that we will have to consider during the remainder of our research should a problem occur.

After understanding that an antibiotic deminishes yeast cell's growth curve, we began to grow S288C yeast cells in varying amounts of the plant hormone Auxin and Ethanol. During this experiment, we also tested ethanol because of the fact that Auxin needed to be dissolved in Ethanol for us to use it. To start several wells containing dilute S288C that were origionally grown overnight were added to differing amounts of 0.25M Auxin and Ethanol. The first row of wells contained no Auxin or Ethanol to act as a control. Then the two following rows either contained 5ul of 0.25M Auxin or 5ul of Ethanol. And a final row was added which contained a mixture of 4ul of Ethanol and 1ul of 0.25M Auxin. After running this experiment three times, we were able to conclude that the Auxin hormone does not affect S288C yeast growth. As seen in the figures above, Auxin was able to follow the same growth curve that can be seen in the Antibiotic test where no antibiotic was added. Therefore, we can conclude that 0.25M Auxin does not affect S288C cell growth and we were able to move forward in testing Coronatine.

After seeing that Auxin does not affect yeast cells, we needed to test its counterpart, Coronatine, to make sure that our bioorthoginal system could actually work. During this experiment, we tested 0.25M Coronatine that was dissolved in DMSO. However, along with testing Coronatine we needed to test the DMSo as well. To start several wells containing dilute S288C that were origionally grown overnight were added to differing amounts of 0.25M Coronatine and DMSO. The first row of wells contained no 0.25M Coronatine or DMSO to act as a control. Then the two following rows either contained 2.8ul of 0.25M Coronatine or 2.8ul of DMSO. And a final row was added which contained a mixture of 1.4ul of DMSO and 1.4ul of 0.25M Coronatine. After running this experiment three times, we were able to conclude that the Coronatine hormone does not affect S288C yeast growth. As seen in the figures above, Coronatine was able to follow the same growth curve that can be seen in the Antibiotic test where no antibiotic was added. Therefore, we can conclude that 0.25M Coronatine does not affect S288C cell growth and we were able to move forward in designing the BAITswitch.