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- All Subjects: Synthetic Biology
- All Subjects: Purification
- Creators: Vogel, Kathleen
- Creators: Chen, Julian
- Member of: Barrett, The Honors College Thesis/Creative Project Collection
To gain more information about the function of the transmembrane region of hTRPM8, it was expressed in Escherichia coli (E. coli) and purified in detergent membrane mimics for experimentation. The construct contains the S4-S5 linker, pore domain (S5 and S6 transmembrane helices), pore helix, and TRP box. hTRPM8-PD+ was purified in the detergents n-Dodecyl-B-D-Maltoside (DDM), 16:0 Lyso PG, 1-Palmitoyl-2-hydroxy-sn-glycero-3-phosphoglycerol (LPPG), and 14:0 Lyso PG, 1-Myristoyl-2-hydroxy-sn-glycero-3-phosphoglycerol (LMPG) to determine which detergent resulted in a hTRPM8-PD+ sample of the most stability, purity, and highest concentrations. Following bacterial expression and protein purification, hTRPM8-PD+ was studied and characterized with circular dichroism (CD) spectroscopy to learn more about the secondary structures and thermodynamic properties of the construct. Further studies can be done with more circular dichroism (CD) spectroscopy, planar lipid bilayer (BLM) electrophysiology, and nuclear magnetic resonance spectroscopy (NMR) to gain more understanding of how the pore domain plus contributes to the activity of the whole protein construct.
The transition from circular to linear chromosomes in eukaryotes introduced the “end-replication problem” which is the inherent inability of cellular DNA polymerases to completely replicate linear chromosomal ends. Over evolutionary time, eukaryotes evolved “caps” at their chromosomal ends which are DNA protein complexes known as telomeres. Although telomeric DNA does suffer from the incomplete end-replication, the telomerase ribonucleoprotein enzyme was evolved as the dominant and winning solution to this problem in eukaryotes. The protein component of telomerase known as Telomerase reverse transcriptase (TERT), is well conserved across broad eukaryotic groups. In contrast, the RNA component of telomerase, telomerase RNA (TR) is extremely divergent in terms of sequence and length. This presents insurmountable challenges in the identification of novel TR molecules, especially from more distant and previously unexplored eukaryotic groups. Although animal TRs have been identified and studied in detail, the early evolution and origins of animal telomerases remain a mystery. Thus, it is crucial to study telomerases from the earliest ancestors of animals. The Choanoflagellates are a group of free-living unicellular eukaryotes that are deemed to be the closes living relatives of animals. The choanoflagellate M. brevicollis (Mbr) is a model eukaryote used to study the origins of multicellularity. Thus, we determined to purify M. brevicollis telomerase to isolate, sequence and identify the co-purifying TR. Towards achieving this ultimate goal, this study focuses on partially purifying M. brevicollis telomerase via polyethylene glycol (PEG) precipitation. As the first step, reliable and reproducible culture conditions for M. brevicollis were established. Following this, larger scale cell cultures were grown and used for PEG precipitation. Final concentrations of 5%, 10%, and 20% PEG were used. PEG precipitates were resuspended in buffer and quantitated using Bradford assay. PEG precipitated macromolecular complexes were subject to Western blot using custom generated anti-MbrTERT antibodies which revealed a clear band proximal in size to the 75 kDa marker consistent with the 87 kDa putative MbrTERT. This study serves as a launchpad for the identification of MbrTR towards delineating the early evolution of telomerase in animals.
Industries and research utilizing genetically-engineered organisms are often subject to strict containment requirements such as physical isolation or specialized equipment to prevent an unintended escape. A relatively new field of research looks for ways to engineer intrinsic containment techniques- genetic safeguards that prevent an organism from surviving outside of specific conditions. As interest in this field has grown over the last few decades, researchers in molecular and synthetic biology have discovered many novel ways to accomplish this containment, but the current literature faces some ambiguity and overlap in the ways they describe various biocontainment methods. Additionally, the way publications report the robustness of the techniques they test is inconsistent, making it uncertain how regulators could assess the safety and efficacy of these methods if they are eventually to be used in practical, consumer applications. This project organizes and clarifies the descriptions of these techniques within an interactive flowchart, linking to definitions and references to publications on each within an Excel table. For each reference, variables such as the containment approach, testing methods, and results reported are compiled, to illustrate the varying degrees to which these techniques are tested.
Industries and research utilizing genetically-engineered organisms are often subject to strict containment requirements such as physical isolation or specialized equipment to prevent an unintended escape. A relatively new field of research looks for ways to engineer intrinsic containment techniques- genetic safeguards that prevent an organism from surviving outside of specific conditions. As interest in this field has grown over the last few decades, researchers in molecular and synthetic biology have discovered many novel ways to accomplish this containment, but the current literature faces some ambiguity and overlap in the ways they describe various biocontainment methods. Additionally, the way publications report the robustness of the techniques they test is inconsistent, making it uncertain how regulators could assess the safety and efficacy of these methods if they are eventually to be used in practical, consumer applications. This project organizes and clarifies the descriptions of these techniques within an interactive flowchart, linking to definitions and references to publications on each within an Excel table. For each reference, variables such as the containment approach, testing methods, and results reported are compiled, to illustrate the varying degrees to which these techniques are tested.
Industries and research utilizing genetically-engineered organisms are often subject to strict containment requirements such as physical isolation or specialized equipment to prevent an unintended escape. A relatively new field of research looks for ways to engineer intrinsic containment techniques- genetic safeguards that prevent an organism from surviving outside of specific conditions. As interest in this field has grown over the last few decades, researchers in molecular and synthetic biology have discovered many novel ways to accomplish this containment, but the current literature faces some ambiguity and overlap in the ways they describe various biocontainment methods. Additionally, the way publications report the robustness of the techniques they test is inconsistent, making it uncertain how regulators could assess the safety and efficacy of these methods if they are eventually to be used in practical, consumer applications. This project organizes and clarifies the descriptions of these techniques within an interactive flowchart, linking to definitions and references to publications on each within an Excel table. For each reference, variables such as the containment approach, testing methods, and results reported are compiled, to illustrate the varying degrees to which these techniques are tested.