Team:CSU Fort Collins/Contribution
Measurement
Project Design
In order to design a plasmid that would allow for further experiment with our quorum sensing circuit, we needed to determine the plasmid with the strongest promoter that could express red fluorescent protein (RFP). We also used Nile red and silica beads to calibrate the plate reader and normalize our results, so that our results could be communicated across all other plate readers. Nile red was decided upon, instead of the fluorescent proteins provided in the measurement kit, because it was available in the lab we were working in, and it was compatible with RFP. The other reason to do an experiment that measured the fluorescence and optical density (OD) is to further characterize parts of the iGEM registry.
The expected results were that the strongest promoter would have the highest fluorescence, and that would be the plasmid J23100. We decided to test three other plasmid constructs provided by iGEM, these were J23104, J23105, J23114. We decided on theses constructs because combined with J23100, there was a wide range of promoter strength. The following table describes this:
| Construct | Promoter Strength |
|---|---|
| BBa_J23100 | 1 |
| BBa_J23104 | 0.72 |
| BBa_J23105 | 0.24 |
| BBa_J23114 | 0.1 |
The construct that we were using in our plasmid design was BBa_J23100, because it was characterized with the strongest promoter strength, and therefore would fluoresce the most.
The design of our experiment was to have four constructs, which were transformed into commercial chemically competent E. coli cells. Then after letting the cells grow on agar plates, four colonies were cultured from each plasmid construct. These colonies were overnight cultured and then diluted down to OD of 0.1. Then each colony from each construct was loaded into the 96 well plate. Another step was to do two serial dilutions of silica beads and Nile red. The silica beads were intended to be used to calibrate the OD and the Nile read was for calibrating and normalizing the fluorescence.
Methods and Material
The protocol that was designed and followed for this experiment is connected to our lab notebookon benchling.
We also used the following protocols from iGEM:
Results and Discussion
Highest Level of Fluorescence
The expected results were that the construct of BBa_J23100 would have the highest fluorescence. That was confirmed with our raw data. There were a few trends that were observed with respect to the fluorescence over time as well as the optical density. For every construct the trend for the fluorescence was that it increases over time, which was reasonable because the more growth that is happening, the more the promoter will be expressed. Since our experiment was over the course of six hours there were a couple trends that we did not see with respect to OD. The typical OD curve that we expected to see was that through the stationary growth phase, but the experiment was only run for six hours, so the cells only reached the very beginning of the stationary growth phase. Because of this the calibration of the OD was not able to be completed.