The Chemical and Biological Engineering Department is pleased to present student seminars by Tracey Dinh and Holly Ekas as part of our ChBE Seminar Series.

Tracey Dinh will present a seminar titled "Forming C-C Bonds via Promiscuous Thiamine Diphosphate Dependent Enzymes."

ABSTRACT: Carbon-carbon bonds make up the backbone of all organic molecules, and its formation is a key reaction step in the synthesis of large, complex compounds. Traditional chemical synthesis routes toward C-C bond formation often require multiple steps for protection and de-protection of non-reacting functional groups. Alternatively, biocatalysts can be implemented to achieve highly specific, single step carboligation due to their inherent regio- and chemoselectivity. A variety of enzymes catalyzing carboligation have been discovered in native biological metabolism; however, their implementation in organic synthesis has been limited due to limited characterization of non-native activity, also known has enzyme promiscuity. In my PhD work, I explored the promiscuity landscape of thiamine diphosphate (ThDP)-dependent enzymes for carboligation. I first demonstrate a platform to comprehensively characterize enzyme substrate promiscuity for bimolecular carboligation reactions. Diverse sets of ?-keto acid substrates were screened in multiplexed reactions, and the resulting products were detected by liquid chromatography mass spectrometry (LC-MS) to generate high-quality enzyme activity data. This screening assay has allowed me to generate the largest database of promiscuous carboligation reactions. Leveraging this bioactivity data, I built cheminformatics-based classifiers to profile enzyme promiscuity and predict activity on native E. coli metabolites. These predictions allow us to identify the potential cell burden due to heterologous expression of promiscuous enzymes.

Holly Ekas will present a seminar titled "Developing high-throughput platforms for cell-free biosensor engineering."

ABSTRACT: Rapid urbanization, outdated infrastructure failures, and agricultural runoff have significantly contaminated our water sources, particularly with heavy metals, fueling the global health crisis in water security. While state-of-the-art analytical chemistry methods provide accurate and sensitive detection of water contaminants, they are expensive, slow, and centralized to laboratories leading to poor accessibility. Cell-free biosensors offer an exciting possibility for on-demand detection of contaminants as they can be cheaply assembled, compared to current commercial tests, and freeze-dried for non-cold-chain transportation. As opposed to cell-based biosensors, cell-free biosensors require minimal equipment and eliminate biocontainment concerns. Allosteric transcription factors (aTF) are an attractive class of protein-based biosensors that rely on using the aTF as a sensor module that interacts with the molecule to be detected to activate or repress transcription of a reporter gene. Most aTFs that sense contaminants of interest to water quality (e.g., heavy metals) that could be used as biosensors are mined from existing bacterial genomes. However, these aTFs are suited for the needs of the bacteria they are derived from and are often not aligned to work optimally in E. coli lysate systems with desired limits of detection, analyte specificity, and dynamic range to be relevant for diagnostic application. In this work, we engineer and optimize the aTF biosensor PbrR for on-demand water quality diagnostics using cell-free expression systems. To do this we developed a screening platform that leverages liquid robotics to identify aTFs with improved characteristics from a library of mutational variants. We found that point mutations can significantly alter aTF promiscuity for detectable analytes as well as dynamic range. We expect that this platform will accelerate future aTF engineering as well as yield biosensors with the limit of detection and dynamic range suited to point-of-use applications.

Bagels and coffee will be provided at 9:30am, and the seminar will start at 9:40am. Please plan to arrive on time to grab a bagel and mingle!

*Please note that there will be no Zoom option for seminars this year.