The goal of this project is to select the variants of an archaea leucyl-tRNA synthetase (MLRS) to incorporate N-𝜀-acetyl lysine (AcLys) into specific positions of proteins in bacterial cells. Acetylation of lysine is one of the most important post-translational modifications of proteins that regulate their functions. One application of this study is using site-directed incorporation of AcLys to introduce novel functions to proteins. Previously, we successfully randomized five positions in the MLRS active site to generate millions of different variants. Genetic screening procedures were performed to select MLRS variants specific for AcLys. Positive selection is performed in the presence of AcLys where bacterial cells containing MLRS that attach any natural amino acids or AcLys onto the tRNA can survive in the presence of chloramphenicol antibiotics. In the negative selection performed in the absence of AcLys, bacterial cells containing MLRS that attach natural amino acids will die in the presence of 5-FU as a toxic substance is produced. Only cells containing MLRS variants that attach AcLys can survive in the presence of 5-FU, because no toxic substance is produced. Two clones made it through multiple rounds of selection and are being tested for successful incorporation of AcLys at the 7th position of the Z-domain protein. Mass spectrometry will be used to detect the presence of AcLys.

Impact of Sensor Design on Hydrogel-Porous Silicon Structures Capable of Detecting Ion Concentrations in Human Sweat

Dylan Walters1, George Weimer1, Leigh T. Canham,2 and Jeffery L Coffer1

1Department of Chemistry and Biochemistry, Texas Christian University, Fort Worth, TX 76129
2Nanoscale Physics, Chemistry and Engineering Research Laboratory, University of Birmingham, Birmingham, B15 2TT UK

Utilizing the supportive structure of hydrogels, the semiconducting character of porous silicon (pSi) membranes, and the biodegradability of both, a unique biosensor for the chemical analysis of health-relevant analytes can ideally be created.
Hydrogels are water-infused, biodegradable polymer networks. Alginate based hydrogels are particularly useful because of environmental abundance, along with their ability to interface well with human skin. The addition of acrylamide segments to the polymer chains adds stability and useful shelf-life to the material. These characteristics also make them an ideal medium for supporting pSi membranes and simultaneously assimilating them into a wide range of tissues.
Porous silicon (pSi), a highly porous form of the elemental semiconductor, is utilized to measure and conduct electrical signals throughout the hydrogel matrix. In diode form, these membranes exhibit measurable current values as a function of voltage, which can be used to detect bioelectrical stimuli such as the concentration of physiologically relevant ionic species (e.g. Na+, K+, and Ca2+).
Recent experiments center on integrating pSi membranes in Acrylamide/alginate co-polymer hydrogels to test how variations in ion concentration affect the flow of current as a function of applied voltage. pSi membranes ~110 m thick and 79% porosity are fabricated from the anodization of low resistivity (100) Si in methanolic HF at an applied bias of 100 mA/cm2 for 30 min. Membrane pieces ~ 2 mm by 2 mm are heated for one hour at 650°C. They are then fashioned into diodes upon the attachment of Cu wire using Ag epoxy and annealed for 15 minutes at 95°C. The backs of the membranes, the connection to the copper wire, and the copper wire itself are sealed using clear nail polish to prevent current flow from the back of the membranes and bubble formation. In each ion sensing experiment, an electrochemical cell is created by placing two pSi membranes parallel each other ~2 mm apart vertically in a fixed electrolyte composition. Current is measured as a function of applied voltage (typically from 0-5 V) for systems with different NaCl concentrations in the nM to mM range. NaCl solutions are injected directly into the hydrogel in between the two pSi membranes 2 µL at a time. At local concentrations of approximately 0.25M, the magnitude of maximum current response increases with increased volume of ion solution added.
This presentation will focus on the porous silicon hydrogel fabrication protocol, as well as results from experiments with varying NaCl concentrations. Future work is being designed to determine the saturation behavior and the ion concentration limits of the pSi membranes in hydrogels.

Fluorescent small molecule environment-sensitive probes change their emission properties (including emission wavelength, intensity or lifetime) in response to the changes of the environment around them, such as changes in temperature, viscosity, and polarity. Thus, these probes have found numerous applications in sensing and imaging, especially in biologically relevant systems. Ratiometic probes is a special group of molecules that has two or more emission wavelengths that exhibit a relative change in response to changes in the media, which provides an internal calibration, increases signal-to-noise ration, and improves the integrity of sensing. However, synthesis of such molecules is usually non-modular in nature, and it often requires multiple steps coupled with numerous purifications. In this presentation, we will highlight our synthetic efforts on the developments of several types of fluorescence ratiometric probes that are based on versatile fluorescence scaffolds, such as BODIPY and squaraine dyes.

Historically, pharmaceutical companies have created small molecule drugs designed to interfere with chemical reactions. An alternative strategy for therapy relies on inhibiting protein-protein interactions, but larger molecules are required. Nature uses large ring-shaped molecules (macrocycles) to accomplish this task. These molecules present challenges to synthesis: forming rings typically is difficult, expensive, time-consuming and inefficient. In addition, the rules required to make macrocyclic drugs are poorly understood when compared to those for small molecules. Here, a strategy for creating macrocycles is described that addresses the challenges of synthesis: they can be prepared quickly and inexpensively. The basis for this chemistry is stepwise substitution of cyanuric chloride, allowing the target to be prepared in three steps. The advantage of using highly electrophilic molecules like cyanuric chloride is that virtually any primary or secondary amine or amino acid could be used to make a macrocycle. Using a variety of amines has shown to affect properties like hydrophobicity and size, which allows for the creation of a large library of molecules to be tested for biological activity, which mirrors how current drug development programs work. The macrocycle is characterized by NMR spectroscopy and screened for other physical (drug-relevant) properties, such as logP and pKa.

FrogCrew is a comprehensive web-based system designed to simplify the management of TCU Athletics sports broadcasting crews. Traditional manual methods of scheduling, tracking availability, and assigning roles are inefficient and prone to errors. This often leads to miscommunication and scheduling conflicts. To solve these challenges, FrogCrew provides a unified platform for administrators. It enables them to manage game schedules, assign roles based on availability and qualifications, and automate notifications efficiently. Key features include customizable crew member profiles. These profiles allow users to update essential information such as availability, roles, and qualifications. The system also offers an automated scheduling tool that simplifies the process of creating game schedules and assigning roles. Additionally, FrogCrew includes a shift exchange feature. This feature allows crew members to request shift swaps, with automated notifications sent to administrators for approval. The system's reporting tools provide financial reports, position-specific insights, and individual performance analyses. These tools help administrators assess crew utilization and manage costs effectively. By automating core functions, FrogCrew reduces manual workload and minimizes errors. It also improves communication between administrators and crew members, ensuring optimal staffing - ultimately enhancing the execution of our TCU sporting events; Go Frogs!