Difference between revisions of "Team:FSU/Results"
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| − | <p>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 | + | <p>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/80 dB, 8 kHz/100 dB, and 2 kHz/100 dB sound. Red fluorescence was measured using a fluorospectrophotometer with excitation of 575 nm and emission of 610 nm. </p> |
| − | <p>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 | + | <p>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 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. |
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| − | + | 8 kHz/80 dB appeared to increase the level of fluorescence in cells with PbamE and K112400. Fluorescence decreased in all other cells. On the other hand, cells exposed to 8 kHz/100 dB sound decreased fluorescence in all cells except those carrying the PosmC and BBa_K112407 promoters. For detailed experimental data see Table 2 and Table 3. | |
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| − | + | 2 kHz/100 dB sound was found to increase the level of fluorescence when compared to ambient sound in all cells containing Berkeley and FSU promoters cells except BBa_K112407. | |
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| − | + | We understood that our project was high risk. We characterized three promoters in the BIOFAB collection (apFAB46, apFAB82 and apFAB90) by measuring their RFP expression level using a fluorospectrophotometer with excitation-emission of 575 and 610 nm. The promoter apFAB46 showed the lowest activity, at 15,819 Mean RFU/OD700, ApFAB90 was found to have medium expression at 28,510 RFU/OD700 and apFAB82 showed the highest expression at 47,118 RFU/OD700. | |
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Revision as of 06:10, 8 December 2018
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/80 dB, 8 kHz/100 dB, and 2 kHz/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 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.
8 kHz/80 dB appeared to increase the level of fluorescence in cells with PbamE and K112400. Fluorescence decreased in all other cells. On the other hand, cells exposed to 8 kHz/100 dB sound decreased fluorescence in all cells except those carrying the PosmC and BBa_K112407 promoters. For detailed experimental data see Table 2 and Table 3. 2 kHz/100 dB sound was found to increase the level of fluorescence when compared to ambient sound in all cells containing Berkeley and FSU promoters cells except BBa_K112407. We understood that our project was high risk. We characterized three promoters in the BIOFAB collection (apFAB46, apFAB82 and apFAB90) by measuring their RFP expression level using a fluorospectrophotometer with excitation-emission of 575 and 610 nm. The promoter apFAB46 showed the lowest activity, at 15,819 Mean RFU/OD700, ApFAB90 was found to have medium expression at 28,510 RFU/OD700 and apFAB82 showed the highest expression at 47,118 RFU/OD700.
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 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.