2024 AIChE Annual Meeting

Effects of Varying Rapamycin Concentrations and Timing on Cellular Functions in Yeast Surface Display

Rapamycin is a commonly used reagent in cell and molecular biology. One use is chemically inducible dimerization. In FKBP-FRM dimerization, rapamycin binds to FKBP (FK506 Binding Protein 12) which it has a high affinity for. It will bind FKBP to the FRB (FKBP Rapamycin Binding) domain of mTOR and control their interactions. These two domains can then be fused to proteins of interest whose interaction the researcher might want to control. However, rapamycin also has direct effects on the cell. It inhibits the mTOR pathway, slowing cellular metabolism. This inhibition of the mTOR pathway is useful in controlling protein synthesis, cell growth and potentially aging, but in the context of controlling synthetic biology, components would yield off-target pleiotropic effects in engineering applications. Therefore, our goal is to determine what concentrations of rapamycin can be used without inhibiting cell growth, gene expression, and other potentially useful functions such as the display of proteins on the surface of yeast.

The experimental platform we used was a yeast surface display system. In our previously developed system (Waldman et al., 2021), we utilize a yeast surface display system that uses a bidirectional galactose promoter. This allows for well-regulated exogenous gene expression of the desired proteins. Specifically, we are transcribing p300 (with a V5 tag) and histone proteins, which include the N-terminus histone tail. P300 is a histone acetyltransferase enzyme that acetylates the lysine residue of histone tails. Aga 2 binds the desired proteins to the surface of our yeast. Through this system, we can detect the display of histone tails, p300 (through a V5 tag), and the occurrence of acetylation between the enzyme and substrate. This detection will be done through the use of two antibodies. The first antibody binds to the desired protein. The second antibody will attach to the first antibody and utilize fluorescence to be detected through flow cytometry. We ran two experiments that tested the effect of rapamycin on this synthetic biology system.

The first experiment tested rapamycin's effect on cell growth in yeast. We tested at different concentrations of rapamycin (0 ng/mL-5000 ng/mL). By adding rapamycin during the growth phase, substrate and enzyme expression were affected, but acetylation (enzymatic function) was not. This also shows that the galactose promoter was inhibited. The second experiment tested rapamycin at different concentrations over different time intervals throughout rapamycin’s induction phase (where gene expression of the plasmid occurs). When rapamycin was added at the same time as the galactose induction, rapamycin greatly attenuated the amount of displayed substrate and enzyme produced. If added 3 hours (or more) after induction, it had almost no effect on substrate and enzyme production. These results could show that all of the displayed substrate is coming from the first 3 hours of induction due to the complete binding of Aga 2. Ongoing analysis will deepen our understanding as to why substrate and enzyme expression is reduced when rapamycin is added at the same time as the galactose promoter was induced.