Difference between revisions of "Team:FSU/Results"

 
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<p>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 —). </p>
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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 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>
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<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.  We measured fluorescence from the cells that contained the Berkeley and FSU promoters.  3 out of the 5 Berkeley promoters (BBa_K112400, BBa_K112401, AND BBa_K112402) and 1 out of the 3 FSU promoters (PpspA) demonstrated basal gene expression when incubated with exposure to ambient sound.  The results are summarized in Figure 1 and 2.
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The cells that contained BBa_ K112400 and PbamE appeared to increase fluorescence when exposed to 8 kHz/80 dB sound.  Fluorescence decreased in all the other cells.  Cells exposed to 8 kHz/100 dB sound decreased fluorescence in all cells except those carrying the PosmC and BBa_K112407 promoters.  Exposing the cells to 2 kHz/100 dB sound correlated with increased levels of fluorescence in all cells except cells that contain BBa_K112407.
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We understood that our project was high risk.  We decided to build and test cells that contained three promoters from the BIOFAB collection (apFAB46, apFAB82 and apFAB90).  by measuring their RFP expression level using A fluorospectrophotometer with excitation-emission of 575 and 610 nm was used to measure red fluorescence. The promoter apFAB46 showed the lowest strength, at 15,819 Mean RFU/OD700, ApFAB90 was found to have medium strength at 28,510 RFU/OD700 and apFAB82 showed the highest strength at 47,118 RFU/OD700.
 
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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 —-).
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<img src="https://static.igem.org/mediawiki/2018/7/7b/T--FSU--berkeley-fsu-promotors.png" style="width:100%">
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<p>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.
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<img src="https://static.igem.org/mediawiki/2018/0/01/T--FSU--berkeley-ambient-sound.png" style="width:100%">
 
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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 —-.
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Figure 2. Berkeley and FSU promoters. Y-axis shows RFU/OD700 of cells in stationary phase and cells grown under ambient sound.
 
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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——.
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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.
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Discussion
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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. In general, we found that cell growth was most aided by 2khz at 100db. On the other hand, cell growth was most disrupted by 8khz and 100db. A frequency of 8khz may be killing cells and thus preventing growth. The power contained in a sound wave of a higher frequency may put too much stress on the cell. 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.
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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.
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<p> 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. </p>
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<img src="https://static.igem.org/mediawiki/2018/7/72/T--FSU--BIOFAB-Promoters-Performance.png" style="width:100%">
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<p> Figure 4. BioFAB promoters performance  On the y-axis, Mean RFU/OD, on the x-axis, promoter used. </p>
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<h2>Discussion</h2>
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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.
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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).
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Table 1. Fluorescence and OD700 of Berkeley and FSU Promoter Test Cells
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<p>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.  </p>
 
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<p> 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.  </p>
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<p>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. </p>
 
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<p> Table 2. Experimental data for 8khz and 80db, 8khz and 100db, and 2khz and 80db. </p>
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<p>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.  </p>
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<p> Table 3. Experimental data for 8khz and 80db. </p>
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<p> Table 4. Experimental data for 8khz and 100db. </p>
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<p> Table 5. Experimental data for and 2khz and 100db. </p>
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Latest revision as of 06:56, 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. We measured fluorescence from the cells that contained the Berkeley and FSU promoters. 3 out of the 5 Berkeley promoters (BBa_K112400, BBa_K112401, AND BBa_K112402) and 1 out of the 3 FSU promoters (PpspA) demonstrated basal gene expression when incubated with exposure to ambient sound. The results are summarized in Figure 1 and 2.

The cells that contained BBa_ K112400 and PbamE appeared to increase fluorescence when exposed to 8 kHz/80 dB sound. Fluorescence decreased in all the other cells. Cells exposed to 8 kHz/100 dB sound decreased fluorescence in all cells except those carrying the PosmC and BBa_K112407 promoters. Exposing the cells to 2 kHz/100 dB sound correlated with increased levels of fluorescence in all cells except cells that contain BBa_K112407.

We understood that our project was high risk. We decided to build and test cells that contained three promoters from the BIOFAB collection (apFAB46, apFAB82 and apFAB90). by measuring their RFP expression level using A fluorospectrophotometer with excitation-emission of 575 and 610 nm was used to measure red fluorescence. The promoter apFAB46 showed the lowest strength, at 15,819 Mean RFU/OD700, ApFAB90 was found to have medium strength at 28,510 RFU/OD700 and apFAB82 showed the highest strength 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 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.