Team:FSU/Results

Results

Each of the Berkeley promoters (BBAK112400, BBAK112401, BBAK112402, BBAK112405 and BBAK112407) and the FSU promoters (BAME, PSPA, and POSMC) were inserted into test devices as outlined in the design section of our project and then exposed to sound conditions. The cells were grown at 37C degrees under ambient sound, 8khz and 80db, 8khz and 100db, and 2khz and 100db and RFP expression level was measured using a spectrofluorophotometer with excitation-emission of 575 and 610nm. Each of the test devices for each of the promoters responded differently under different sound conditions (See Figure —).

The first phase of the experiment consisted of growing the cells in an incubator at 37 degrees for 8 hours as a control measure. Growth of Berkeley and FSU cells was compared against a positive control, which was a constitutive promoter, and a negative control, a strain of NEB-5-alpa that had no RFP expression. Results showed that of the tested cells, 3 out of the 5 Berkeley promoters (BBAK112400, BBAK112401 AND BBAK112402) and 1 out of the 3 FSU promoters (PSPA) demonstrated baseline expression in the absence of any sound induction. In order to correct for the baseline expression of RFP in the absence of sound of some strains, data is shown as a ratio of RFU/OD700 (See Figure —-).

A frequency of 8khz at 80db increased the level of fluorescence only in PBAME and K112400 promoter cells and marginally decreased the level of fluorescence in all other cells. On the other hand, 8khz at 100db down regulated the expression of RFP in all cells except those carrying the POSMC and K112407 promoters. For detailed experimental data see Table — and Table —-.

A frequency of 2khz and 100db was found to increase the level of fluorescence when compared to ambient sound in all cells except K112407 promoter cells. The FSU iGEM promoters: BamE, OsmC, and PspA also showed an increase in fluorescence after sound exposure see Figure—- and Table——.

Because we acknowledged that our project was high risk, we characterized three of the promoters in the BIOFAB collection, namely, apFAB46, apFAB80 and apFAB90 by measuring their RFP expression level using a spectrofluorophotometer with excitation-emission of 575 and 610nm. The promoter apFAB46 showed the lowest activity, at 15,819 Mean RFU/OD, ApFAB90 was found to have medium expression at 28,510 RFU/OD and apFAB82 showed the highest expression at 47,118 RFU/OD.

Discussion

In general, we found that cell growth was most aided by 2khz at 100db and most disrupted by 8khz and 100db. A frequency of 8khz may represent a mechanical stress too strong for the cell to be able to grow under. We found that two of the Berkeley promoters had components of constitutive expression since they showed significant fluorescence after growing under ambient sound. The power contained in a sound wave of 8khz may be too high and could be causing too much stress on the cells. Based on the data obtained, it is possible that a sound wave at 2khz is able to induce a mechanical stress response that activates the systems in some of our promoter test cells without causing harm.

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. Since unexpected levels of promoter activities were seen, this may be an indication that the performance of BIOFAB parts may be influenced by specific growth conditions and not only on ribosome binding sites like previously thought.

Figure 1. Berkeley and FSU promoters. Y-axis shows RFU/OD700 of cells in stationary phase and cells grown under ambient sound.


Figure 2. Berkeley and FSU Promoters. Y-axis shows ratio of RFU/OD of stationary phase cells to cells grown under ambient sounds, 8khz 100db, 8khz 80db, and 2khz 100db.



Figure 3. Berkeley and FSU Test Cells After Incubation in Ambient Sounds y-axis shows RFU/OD700 and x-axis shows the promoter that each cell line carries.


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

Figure 4. Statistical data for Fluorescence and OD700 OF Berkeley Test Cells and FSU promoters


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.