Team:FSU/Results
Results
Although the Berkeley Test Cells showed an increase in fluorescence when exposed to sound. Further testing must be done to further characterize this initial response to sound. The Berkeley promoter BBa_K112402 was found to be a constitutive promoter. The same promoter, BBa_K112402, also shows a possible RFP upregulation when exposed to sound however further testing needs to be done to determine a more detailed relationship between amplitude, frequency and promoter upregulation. The FSU iGEM promoters: BamE, OsmC, and PspA also showed an increase in fluorescence after sound exposure.
In addition to characterizing the promoters proposed by UC Berkeley in 2008, we wanted to bring IGEM the BIOFAB collection of promoters. We picked three promoters, ApFAB90, ApFAB46, and ApFAB82. The activity of the promoters was characterized by inserting them into a PSCB1C3 plasmid which contained a red cassette that expresses RFP. We measured activity by using a ratio of Fluorescence to optical density and comparing initial and final values. We expected each promoter to behave with low, medium, and high activity respectively. The BIOFAB promoter behavior was not in the order that we were expecting. The apFAB46 showed the lowest activity but was expected to be the highest. ApFAB90 was found to have medium expression, however, was expected to be low activity. Finally, apFAB82 showed the highest fluorescence, however, was expected to be the medium expression. Further investigation is needed to determine these unexpected results.
| Cells | Mean RFU/OD700 | Standard Deviation | Mean RFU | Standard Deviation | Mean OD700 | Standard Deviation |
|---|---|---|---|---|---|---|
| Negative Control | 81 | 11.8 | 49 | 6.7 | 0.601 | 0.011 |
| Positive Control | 28642 | 2781.7 | 12028 | 854.2 | 0.425 | 0.074 |
| BBa_K112400 | 12872 | 250.6 | 11993 | 425.0 | 0.932 | 0.026 |
| BBa_K112401 | 2861 | 41.4 | 3082 | 164.7 | 1.077 | 0.057 |
| BBa_K112402 | 24572 | 837.7 | 20455 | 507.1 | 0.833 | 0.014 |
| BBa_K112405 | 304 | 11.3 | 246 | 12.6 | 0.807 | 0.013 |
| BBa_K112407 | 148 | 0.5 | 131 | 2.5 | 0.882 | 0.016 |
| PosmA | 190 | 10.5 | 85 | 2.0 | 0.449 | 0.036 |
| PpspA | 828 | 46.2 | 371 | 9.7 | 0.449 | 0.030 |
| PbamE | 165 | 16.2 | 73 | 7.2 | 0.441 | 0.010 |
| PzntA | 168 | 26.1 | 73 | 5.6 | 0.439 | 0.036 |
Using a constitutive promoter as a positive control, we compared the flourescence/OD700 of the Berkeley promoters when allowed to grow in the incubator with “ambient sound”. We found that the expression of BBa_K112402 was comparable to he positive control. We believe this might be an indication that BBa_K112402 is a constitutive promoter. BBa_K112400 also had a high expression and we believe that this may indicate that it may also be a weaker constitutive promoter.
The BIOFAB promoters showed an unexpected level of activity. The promoter apFAB46 showed the lowest activity but was expected to be the highest. ApFAB90 was found to have medium expression, however, was expected to be low activity. Finally, apFAB82 showed the highest fluorescence, however, was expected to be the medium expression.
We found that two of the Berkeley promoters appeared to be constitutive promoters since they showed significant fluorescence after growing under ambient sound. The PSPA FSU system was likely slightly leaky since it shows a slight trace of fluorescence under ambient sound.
After exposing our different test cells to different frequencies and amplitudes, we found that cells did seem to respond differently to different sonic stress conditions based on the promoters they carried. We fund that cell growth was most aided by 2khz at 100db. On the other hand, cell growth was most disrupted by 8khz and 100db, and we think this frequency may actually be killing cells and thus preventing growth. In general, the PSPA system was the most sound responsive system and K112402 was likely a constitutive promoter.
