Search for: All records

Transcription is the critical first step in expressing a gene, during which an RNA polymerase (RNAP) synthesizes an RNA copy of one strand of the DNA that encodes a gene. Here we describe a laboratory experiment that uses a single assay to probe two important steps in transcription: (1) RNAP binding to DNA, and (2) the transcriptional activity of the polymerase. Students probe both these steps in a single experiment using a fluorescence‐based electrophoretic mobility shift assay (EMSA) and commercially availableEscherichia coliRNAP. As an inquiry‐driven component, students add the transcriptional inhibitor rifampicin to reactions and draw conclusions about its mechanism of inhibition by determining whether it blocks polymerase binding to DNA or transcriptional activity. Depending on the curriculum and learning goals of individual courses, this experimental module could be easily expanded to include additional experimentation that mimics a research environment more closely. After completing the experiment students understand basic principles of transcription, mechanisms of inhibition, and the use of EMSAs to probe protein/DNA interactions.

The secretome from mesenchymal stem cells (MSCs) have recently gained attention for new therapeutics. However, clinical application requires in vitro cell manufacturing to attain enough cells. Unfortunately, this process often drives MSCs into a senescent state that drastically changes cellular secretion activities. Antioxidants are used to reverse and prevent the propagation of senescence; however, their activity is short‐lived. Polymer‐stabilized crystallization of antioxidants has been shown to improve bioactivity, but the broad crystal size distribution (CSD) significantly increases the efficacy variation. Efforts are made to crystalize drugs in microdroplets to narrow the CSD, but the fraction of drops containing at least one crystal can be as low as 20%. To this end, this study demonstrates that in‐drop thermal cycling of hyaluronic acid‐modified antioxidant crystals, named microcrystal assembly for senescence control (MASC), can drive the fraction of microdrops containing crystals to >86% while achieving significantly narrower CSDs (13 ± 3 µm) than in bulk (35 ± 11 µm). Therefore, this approach considerably improves the practicality of CSD‐control in drops. In addition to exhibiting uniform release, MASC made with antioxidizing N‐acetylcysteine extends the release time by 40%. MASC further improves the restoration of reactive oxygen species homeostasis in MSCs, thus minimizing cellular senescence and preserving desired secretion activities. It is proposed that MASC is broadly useful to controlling senescence of a wide array of therapeutic cells during biomanufacturing.