Tilbury, Hofherr, Boyea, McDonald, Schnack, Kuhlmann, Soltesz/URF25/Miami University

GG/CC/gtCC

Motivation

Our goal was to change the secondary binding site at the -10, as seen highlighted by the second red line in the photo below. The purpose of this was to block the natural sigma factor while still allowing for the binding of ECF 11. Thus, we only had two base pairs, gt, which we could edit without affecting the binding site for the ECF 11. We decided to break the unwanted binding site by changing gt to GG and CC and tried another method where we kept the gt and inserted a CC afterwards, creating gtCC. All of these were evaluated at a 12 bp, 13 bp and 16 bp length. We wanted to have no fluorescence in the 12 bp with ECF11 and ECF11 empty and only wanted fluorescence in the 13 and 16 bp with ECF 11. The presence of fluorescence means the base pair is active.

Chart showing ideal spacer length for ECF 11

This shows that the preferred spacer length for ECF 11 is 16 base pairs, so 16 base pairs is where we expect the most fluorescence.

Binding site region and translation region used in experiment

This shows the binding site and the translation region that was used for the experiment. The gt that is right before the second red line is what was changed.

GG/CC

The GG/CC approach changed the GT base pair highlighted in the photo with CC and GG, creating a 12bp and 16bp for a total of 4 unique samples. The controls for both CC and GG were the original unedited ECF 11 binding sites and the altered binding sites with no ECF 11 present.

GTCC

We added CC to the GT highlighted in the photo above for 12, 13, and 16bp, creating 9 unique samples. This was hypothesized to have the same success as GG and CC through the software Softberry.

Findings

GG/CC

CC operated as hypothesized, having minimized interaction with E. coli’s natural sigma factors (fig 3, 5), and had little interference with the ECF 11 sigma factor binding site. GG also acted as hypothesized (fig 2,4), the 16bp Sigma factor showed the most fluorescence by a large margin. CC worked better than GG.

GTCC

gtCC operated as hypothesized, with no secondary binding site on the 12, 13 and 16 bp (fig 6, 9), without interfering with the ECF 11 sigma factor binding site (fig 7, 8). Despite being hypothesized to function the same as GG and CC, gtCC was found to have better results than GG and CC.