Throughout the course of our project, many of our experimental plans underwent multiple design cycles. This process began with our plasmid designs. For the FimH plasmids, we needed to not only make sure to use a tag that would have a strong affinity for our marker of interest but also design a control plasmid to ensure accurate measurement of tag affinity. FimH is known to strong affinity to mannose1, so we chose to use a histidine tag due to shown that chimera’s of FimH with a hexahistidine tag could be stimulated by binding to Ni-NTA beads. The SNAP tag can be attached to a protein of interest and functions to tag the targeted antibodies. FimH His and SNAP tags were designed in benchling for our experimental use.
We knew from the literature that FimH was connected the lacZ transcription factor OxyR2, but figuring out the exact connection stumped us. Through additional research and a conversation with Dr. Robert Shanks, we discovered that FimH binding up-regulated OxyR transcription through the genes katG3, dps4,5, and grxA6,7. Plasmids gene reporter assays were designed o regulate the expression lacZα using the Benchling website. We also discover that hypoxia can induce OxyR, so we began to design experiments to test gene transcription level in response to hypoxia akin to that of the tumor microenvironment.
Our H2O2 experiment required the most rounds of trouble shooting. We modeled our determination of critical H2O2 concentration after Rodríguez-Rojas et. al. and our lacZ assay after Schaefer et al.. In our initial run, we did not see any color change. We decided to add lysis buffer to optimally exposed the lacZ to the x-gal solution. However, our efforts still failed, and we had to go back to the drawing board. Through team discussions and guidance from our mentors, we decided to put the experiment on hold until we could determine the critical H2O2 concentration to ensure optimal cell impact. We determined this value to be 1mM. This concentration was subsequently used as a reference for 30-minute killing curves that show a clear dose-effect in survival rate.
We were still finding little success with this experiment, as we did not see a significant difference across varying concentrations of H2O2. We utilized a more involved strategy of sampling from culture tubes rather than our initial method of using a 96 well plate. The 96 well plates did not allow the bacteria to grow optimally due to poor volume of culture and not enough air. We made the cultures from frozen stock, grew them for 30 minutes to reach the exponential phase, then from this phase we started the timer and every 30 minutes took a sample from each of the concentrations of each of the plasmid types (8 samples at each interval total). The final procedure can be found in Experiments.
- Schwan, W. R.; Beck, M. T.; Hung, C. S.; Hultgren, S. J. Differential Regulation OfEscherichia Coli FimGenes Following Binding to Mannose Receptors. Journal of Pathogens 2018, 2018, 1–8. https://doi.org/10.1155/2018/2897581.
- Shanks, R. M. Q.; Stella, N. A.; Kalivoda, E. J.; Doe, M. R.; O’Dee, D. M.; Lathrop, K. L.; Guo, F. L.; Nau, G. J. A Serratia Marcescens OxyR Homolog Mediates Surface Attachment and Biofilm Formation. Journal of Bacteriology 2007, 189 (20), 7262–7272. https://doi.org/10.1128/JB.00859-07.
- Jung, I. L.; Kim, I. G. Transcription of AhpC, KatG, and KatE Genes in Escherichia Coli Is Regulated by Polyamines: Polyamine-Deficient Mutant Sensitive to H2O2-Induced Oxidative Damage. Biochemical and Biophysical Research Communications 2003, 301 (4), 915–922. https://doi.org/10.1016/s0006-291x(03)00064-0..
- Antipov, S. S.; Tutukina, M. N.; Preobrazhenskaya, E. V.; Kondrashov, F. A.; Patrushev, M. V.; Toshchakov, S. V.; Dominova, I.; Shvyreva, U. S.; Vrublevskaya, V. V.; Morenkov, O. S.; Sukharicheva, N. A.; Panyukov, V. V.; Ozoline, O. N. The Nucleoid Protein Dps Binds Genomic DNA of Escherichia Coli in a Non-Random Manner. PLoS ONE 2017, 12 (8), e0182800. https://doi.org/10.1371/journal.pone.0182800.
- Calhoun, L. N.; Kwon, Y. M. Structure, Function and Regulation of the DNA-Binding Protein Dps and Its Role in Acid and Oxidative Stress Resistance in Escherichia Coli: A Review. Journal of Applied Microbiology 2010, 110 (2), 375–386. https://doi.org/10.1111/j.1365-2672.2010.04890.x.
- grxA - Glutaredoxin 1 - Escherichia coli (strain K12) - grxA gene & protein https://www.uniprot.org/uniprot/P68688 (accessed May 24, 2021).
- Prieto-ÁlamoM.-J.; Jurado, J.; Gallardo-Madueño, R.; Monje-Casas, F.; Holmgren, A.; Pueyo, C. Transcriptional Regulation of Glutaredoxin and Thioredoxin Pathways and Related Enzymes in Response to Oxidative Stress. Journal of Biological Chemistry 2000, 275 (18), 13398–13405. https://doi.org/10.1074/jbc.275.18.13398.