Team:FSU/Results

Results

The Berkeley promoters (BBa_K112400, BBa_K112401, BBa_K112402, BBa_K112405 and BBa_K112407) and the FSU promoters (pBamE, pPspA, and pOsmC) were used in test devices described in the design section. The devices were inserted into the pSB1C3 plasmid backbone. The resulting plasmid vector was used to transform E. coli NEB 5-alpha chassis. The engineered cells were grown at 37 degrees celsius exposed to ambient, 8 khz and 80 dB, 8 khz and 100 dB, and 2 khz and 100 dB sound. Red fluorescence was measured using a fluorospectrophotometer with excitation of 575 nm and emission of 610 nm.

The first phase of the experiment consisted of growing the cells in an incubator at 37 degrees for 8 hours exposed to ambient sound. 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 (BBa_K112400, BBa_K112401, AND BBa_K112402) and 1 out of the 3 FSU promoters (pPspA) demonstrated baseline expression in the absence of any sound induction.

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 2 and Table 3.

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 2).

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 fluorospectrophotometer 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.



Figure 1. Berkeley and FSU Test Cells After Incubation in Ambient Sounds. The horizontal axis lists the promoters tested in engineered cells. The vertical axis is Relative Fluorescence Units/OD700.



Figure 2. Berkeley and FSU promoters. Y-axis shows RFU/OD700 of cells in stationary phase and cells grown under ambient sound.



Figure 3. 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 4. BioFAB promoters performance On the y-axis, Mean RFU/OD, on the x-axis, promoter used.



Discussion

The results indicate that BBa_K112400, BBa_K112401, BBa_K112402, and BBa_K2832003 (PpspA) promote a baseline of gene expression that is above the negative control. The results suggests that BBa_K112400, BBa_K112401, BBa_K2832003 (PpspA) increase expression when exposed to 2 kHz/100 dB sound. The cells containing BBa_K112400, BBa_K112401, BBa_K112402, BBa_K112405, and BBa_K2832003 (PpspA) demonstrated a decrease in fluorescence after exposure to 8 kHz sound. We do not know why. Exposing the cells to 8 kHz sound for 8 hours may be harmful or inhibit gene expression.

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 (See Figure 5).

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


Table 1. Fluorescence and OD700 of Berkeley and FSU Promoter Test Cells




Table 2. Experimental data for 8khz and 80db, 8khz and 100db, and 2khz and 80db.


Table 3. Experimental data for 8khz and 80db.


Table 4. Experimental data for 8khz and 100db.


Table 5. Experimental data for and 2khz and 100db.