Photosynthesis Lab Report

The first goal of the lab was to identify conserved sequence segments in the ykkCD sensor RNA of Bacillus subtilis. Toxin sensors that do not change throughout evolution have a substantial role for the cell, even significant roles in tetracycline recognition. Using RNA sequences and computer programs, secondary structures of the ykkCD sensors were created. Figure 1 shows a prediction of the secondary structure of a ykkCD senor RNA of Bacillus subtilis that has been conserved throughout its evolution. Figure 1: The secondary structure prediction for Bacillus subtilis ykkCD sensor RNA.

After identifying the ykkCD sensor, site-directed mutagenesis will be used to mutate the sensor. Mutating the sensor will identify if it is used in tetracycline

Plasmid DNA from E. coli cells was extracted and purified. To extract the plasmid DNA and purify it, the plasmid DNA had to be placed inside the bacterial cell through the process of transformation. The cells that took the plasmid DNA were grow grown in a media containing the antibiotics. Then the plasmid DNA is purified by removing genomic DNA, lipids and membrane proteins. With the DNA template containing the mutant sensor RNA, The DNA template was prepared for RNA synthesis using restriction endonuclease. The mutant senor was then synthesized using the plasmid DNA template and T7 RNA polymerase. The mutant toxin sensor RNA was purified to remove any polymerase, DNA template unused nucleotides and buffers. Finally, the ykkCD toxin sensor to recognize the antibiotic tetracycline was evaluated using Fluorescent Quenching. Figure 3 shows the results of the mutated RNA by graphing the inverse of quenching versus the inverse of the mutated RNA concentration. Figure 4 displays the inverse of quenching versus the inverse of the concentration of the wildtype

To estimate how well the mutated sensor RNA recognizes the antibiotic tetracycline, the binding affinity of the tetracycline-sensor RNA complex (KD) was measured. The binding affiniy is equal to the slope of the graphs. For these graphs the slope of the mutated RNA was 0.2632 and the wildtype RNA was 0.0721. Using the slopes obtained an equation can be used to evaluate the ykkCD sensor RNA mutant. Equation 1 shows the calculation needed to make the estimation of the sensor. 〖K_D〗^Mutant/〖K_D〗^Wildtype
KDMutant is the binding affinity of the mutant sensor while KDWildtype is the binding affinity of the wildtype sensor. If the value is greater than 10, then part of the sensor targeted for mutagenesis was important for tetracycline recognition. If the calculation is below 10, then the sensor was not essential to recognize tetracycline. The calculation for the tetracycline recognition is shown below.
0.2632/0.0721=3.65
Using equation 1 and the slopes of the graphs from figures 3 and 4, a value of 3.65 was obtained which explains that the sensor that was experimented on does not have the ability to recognize