Difference between revisions of "Team:FSU/Results"

 
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<p>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. </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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<p>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.  
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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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<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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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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<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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<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> Table 3. Experimental data for 8khz 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 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.