Our motivation for this lab was to build upon unexplored aspects of the ECF11 sigma factor used in Dr. Boock’s previous classes. Our goal was to find the true binding site of ECF11 and to test the conservation of the base pair CC in the -35 promoter region. Using the sequence logo chart of the natural binding site of ECF11, we observed that the binding site became less conserved at position -31. By mutating this base pair into each of the other nucleotide bases, we hypothesized that the most conserved nucleotide at position -31 would see the highest levels of GFP production.
After inserting the designed binding sites into the plasmids, they were introduced into E. coli through heat shock. We then picked one colony for each binding site tested and subcultured each into three samples for error correction. Each person grew one construct with Sigma factor ECF11 and one without. We also tested the original pSFB p11 16bp GFP binding site to compare with our modified sequences. A negative control group with only LB was included to test for background fluorescence.
When evaluating our hypothesis, we found a few differences between our predictions and our results
C31T/C32T with and without the sigma factor glowed more than predicted
C31A produced more GFP than any of the other samples
C31G and C31T produced similar levels of fluorescence
All samples that included ECF11 produced more fluorescence than the trial using the unmodified binding site
Main Finding: The -31/-32 segment of the -35 binding site binding site for ECF 11 sigma factor not strongly conserved.