Team:RHIT/Results
Results
Results of the Fluorescence Measurements
The first thing to determine with the riboswitches was if they responded properly within our specific cell environment and with our construct. To determine if the riboswitches responded to the addition of the vitamin in the form of Leucovorin Calcium for the B9 Riboswitches or Cyanocobalamin for the B12 Riboswitches, we plotted the normalized fluorescence against the hour for the second set of data. These produced plots like following:
[[File:Fig_1_rhit.jpg]]Figure 1: Aliquots of the T. Tengcongensis riboswitch culture were taken at 0,2,4,8, and 20 hours. The cultures were inoculated at 0 hours into either a solution of M9 minimal or M9 minimal and 0.03 mM of added cyanocobalamin.
Generally after 4-8 hours you can see a significant difference in expression between the assays with no added repressor and that with the repressor. By the 20 hour point, the cells are reducing the expression in both assays, though the expression levels between the two samples remains distinctly different. This reduction in expression per OD630 units is likely due to cell death within the liquid cultures. Because the most distinct difference between the riboswitches occurred 8 hours after the addition of the repressor, we plotted the normalized fluorescence against the concentration of the added ligand in the culture at this time producing the following plots.
*****FIGURE*****The B9 riboswitches showed variable fluorescence expression at the no and low concentration repressor, however after the concentration of Leucovorin Calcium increases to and beyond 0.5 mM, the fluorescence approaches zero comparatively. The B12 has the same trend when the concentration of Cyanocobalamin, but the E. coli riboswitch begins with a much lower fluorescence output. Additionally, the riboswitches potentially are slightly more sensitive, as a much more distinct difference is seen in the 0.03 mM concentration.
***FIGURE***This allows us to see distinct differences in output at different concentrations of the binding repressor and verify the operation of the riboswitches in the constructed parts.
Figure 1. ITC graph using 20 2.49 µL injections of 1.23 M Vitamin B9 solution in 50 mM HEPES buffer into a sample cell containing 175 µL of 0.25 µM B9-1 riboswitch solution in 50 mM HEPES buffer.
Figure 2. Zoomed-in version of Figure 1 excluding the first large negative slope before 300 seconds due to injection 1.
Analysis
The first injection took place at 0 seconds, and as shown in Figure 1, that injection caused a very large heat rate followed by what appears to be no further heat rate induced by the later injections. However, when this graph was zoomed in as shown in Figure 2, there were peaks with each of the following injections. This indicates that the first injection of Vitamin B9 interacted with most of the B9-1 riboswitch present in the sample cell which left the following heat rate peaks to be very small in comparison because there was not enough sample for the titrant to have a comparable reaction with. Therefore, for the next experiment, the titrant concentration will be reduced and tested.
Figure 3. ITC graph using 20 2.49 µL injections of 0.60 µM Vitamin B9 solution in 50 mM HEPES buffer into a sample cell containing 175 µL of 0.25 µM B9-1 riboswitch solution in 50 mM HEPES buffer.
Although the first injection did not react with all of the sample as before and the first 19 samples showed large peaks, there was not a gradual decline of heat rate. Instead, the 20th injection peak had a much smaller magnitude which indicated that the sample ran out just then. This could be due to faulty stoichiometry predictions, non-ideal injection times, and/or still incorrect concentration ratios and will be looked into further.
