<p> </p>
<p>Biomolecular condensates or membrane-less organelles are phase separated proteins and/or other biomolecules that are ubiquitous in eukaryotic cells. While these condensates may be liquid with exchange and diffusion of their components with the rest of the cell (e.g., cytoplasm), they locally concentrate their constituent biomolecules altering their interactions in normal cellular processes. This phenomenon has been exploited for static control of protein activity in <em>E. coli</em>. However, neither dynamic control of protein activity using external triggers nor programmable tuning of protein activity has been explored so far.</p>
<p>To address these gaps, I fused proteins of interest to elastin-like polypeptides (ELP) that aggregate in response to an increase in temperature. In so doing, I sequester their fusion partners from the cytoplasm, limiting their ability to participate in cytoplasmic reactions. I have demonstrated this concept<em> in vivo</em> with enzymes and transcription factors for switchable control of protein activity with temperature. For example, I-SceI mediated cleavage of a host genome can be inhibited by increasing the cultivation temperature, creating a simple temperature-sensitive kill switch; accidental release will lower the culture temperature leading to cell death. Similarly, coupled transcription factors exhibit a 2-fold increase in transcription relative to unfused transcription factor controls at elevated temperatures. More importantly, the threshold for coacervate formation and control of protein activity may be tuned through appropriate design of the ELP used for fusion. Furthermore, the temperature response of the ELP fusion is unique to each protein and depends on the structure of the fusion partner, which dictates the structure of the ELP fused aggregate. Our results introduce a simple yet effective, rapid, and tunable approach to control protein activity via induction of coacervate formation that may form a powerful new tool for synthetic biology.</p>
| Identifer | oai:union.ndltd.org:purdue.edu/oai:figshare.com:article/23535225 |
| Date | 16 June 2023 |
| Creators | Mrugesh Krishna Parasa (16384935) |
| Source Sets | Purdue University |
| Detected Language | English |
| Type | Text, Thesis |
| Rights | CC BY 4.0 |
| Relation | https://figshare.com/articles/thesis/Programmable_Control_of_Protein_Activity_Via_Formation_of_Biomolecular_Condensates_in_Bacteria/23535225 |
Page generated in 0.0021 seconds