Optimization of an in-house bacterial cell-free expression system to evaluate the design of toehold switch sensors for selected cervical precancer miRNA biomarkers

Cervical cancer is a significant health burden for women across the globe. However, over 94% of all cervical deaths occured in low and middle income countries (LMICs) in 2022 alone. This reflects a broader trend of a lack of access to quality treatment options for cervical cancer due to socioeconomic barriers including limited trained professionals, financial resources, and access to proper screening. We are partnering with UMaryland iGEM, an undergradutate-led synthetic biology research team, to assist in the development of a low-cost, point-of-care screening device for detecting commonly upregulated miRNAs in cervical precancer patients to address this issue. The functionality of this device relies on a cell-free expression kit, referred to as a lysate, in order to properly transcribe the desired toehold switches in the paper-based assay and translate the fluorescent protein output. However, common commercial cell-free lysates are expensive which increases the cost of producing our device – ultimately hindering its accessibility in LMICs. To address this problem, we propose a method for producing and optimizing our own in-house cell-free lysate. In our method, we compare three lysates – commercially available myTXTL, a lysate developed by the Aberdeen Proving Ground, and our own lysate – using varying ratios of energy mix, lysate, and T7p14 deGFP HP plasmid to evaluate the ideal composition of these components for a cell-free expression kit. We quantify these relationships using fluorescence in order to determine the efficacy of our lysate and the ideal ratio of components for a cell-free, paper-based assa

Conserved mRNA-granule component Scd6 targets Dhh1 to repress translation initiation and activates Dcp2-mediated mRNA decay in vivo.

Scd6 protein family members are evolutionarily conserved components of translationally silent mRNA granules. Yeast Scd6 interacts with Dcp2 and Dhh1, respectively a subunit and a regulator of the mRNA decapping enzyme, and also associates with translation initiation factor eIF4G to inhibit translation in cell extracts. However, the role of Scd6 in mRNA turnover and translational repression in vivo is unclear. We demonstrate that tethering Scd6 to a GFP reporter mRNA reduces mRNA abundance via Dcp2 and suppresses reporter mRNA translation via Dhh1. Thus, in a dcp2Δ mutant, tethered Scd6 reduces GFP protein expression with little effect on mRNA abundance, whereas tethered Scd6 has no impact on GFP protein or mRNA expression in a dcp2Δ dhh1Δ double mutant. The conserved LSm domain of Scd6 is required for translational repression and mRNA turnover by tethered Scd6. Both functions are enhanced in a ccr4Δ mutant, suggesting that the deadenylase function of Ccr4-Not complex interferes with a more efficient repression pathway enlisted by Scd6. Ribosome profiling and RNA-Seq analysis of scd6Δ and dhh1Δ mutants suggests that Scd6 cooperates with Dhh1 in translational repression and turnover of particular native mRNAs, with both processes dependent on Dcp2. Our results suggest that Scd6 can (i) recruit Dhh1 to confer translational repression and (ii) activate mRNA decapping by Dcp2 with attendant degradation of specific mRNAs in vivo, in a manner dependent on the Scd6 LSm domain and modulated by Ccr4

Preparation and Application of In-House E. coli Cell-Free Protein Expression Kits

Cell-free expression technology is cutting-edge research that would allow for increased flexibility, reliability, and versatility in the development of biosensors. This project is a joint between the Molecular Diagnostics and Cell-Ex labs in the FIRE program at the University of Maryland, in which we examine the usability of cell-free expression for workflows that differ from what they have been designed for, and conduct research into the feasibility of creating our own cell-free extract for further experimentation. In doing so, we determine the feasibility of using cell-free extract across different lab workflows, the possibility of creating it , and the cost-effectiveness of doing so versus commercial alternatives

Development of a Certification Exam to Assess Undergraduate Students\u27 Proficiency in Biochemistry and Molecular Biology Core Concepts

With support from the American Society for Biochemistry and Molecular Biology (ASBMB), a community of biochemistry and molecular biology (BMB) scientist-educators has developed and administered an assessment instrument designed to evaluate student competence across four core concept and skill areas fundamental to BMB. The four areas encompass energy and metabolism; information storage and transfer; macromolecular structure, function, and assembly; and skills including analytical and quantitative reasoning. First offered in 2014, the exam has now been administered to nearly 4000 students in ASBMB-accredited programs at more than 70 colleges and universities. Here, we describe the development and continued maturation of the exam program, including the organic role of faculty volunteers as drivers and stewards of all facets: content and format selection, question development, and scoring

O-GlcNAc Cycling Enzymes Associate with the Translational Machinery and Modify Core Ribosomal Proteins

At least 20 core ribosome proteins are modified by O-GlcNAc. O-GlcNAcase is localized to the nucleolus and O-GlcNAc transferase is excluded from the nucleolus. Both enzymes associate with active polysomes. Overexpression of OGT disrupts ribosomal subunit homeostasis. Data suggest that O-GlcNAc regulates translation and ribosome biogenesis