iGEM 2018 — Team FSU

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# Team:FSU/Notebook

[Home](https://2018.igem.org/Team%3AFSU)
[Description](https://2018.igem.org/Team%3AFSU/Description)
[Human Practices](https://2018.igem.org/Team%3AFSU/Human_Practices)
[Design](https://2018.igem.org/Team%3AFSU/Design)

Build & Test

[Experiments](https://2018.igem.org/Team%3AFSU/Experiments)
[Results](https://2018.igem.org/Team%3AFSU/Results)
[Improve](https://2018.igem.org/Team%3AFSU/Improve)
[Part Collection](https://2018.igem.org/Team%3AFSU/Part_Collection)
[Parts](https://2018.igem.org/Team%3AFSU/Parts)

Attributions

[Team & Attributions](https://2018.igem.org/Team%3AFSU/Attributions)
[Collaborations](https://2018.igem.org/Team%3AFSU/Collaborations)
[References](https://2018.igem.org/Team%3AFSU/References)

[Judging Form](https://igem.org/2018_Judging_Form?team=FSU)
☰

# Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

### What should this page have?

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

### Inspiration

You can see what others teams have done to organize their notes:

* [2014 ATOMS-Turkiye](https://2014.igem.org/Team%3AATOMS-Turkiye/Notebook)
* [2014 Tec Monterrey](https://2014.igem.org/Team%3ATec-Monterrey/ITESM14_project.html#tab_notebook)
* [2014 Kyoto](https://2014.igem.org/Team%3AKyoto/Notebook/Magnetosome_Formation#title)
* [2014 Cornell](https://2014.igem.org/Team%3ACornell/notebook)


[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:FSU/Experiments

[Home](https://2018.igem.org/Team%3AFSU)
[Description](https://2018.igem.org/Team%3AFSU/Description)
[Human Practices](https://2018.igem.org/Team%3AFSU/Human_Practices)
[Design](https://2018.igem.org/Team%3AFSU/Design)

Build & Test

[Experiments](https://2018.igem.org/Team%3AFSU/Experiments)
[Results](https://2018.igem.org/Team%3AFSU/Results)
[Improve](https://2018.igem.org/Team%3AFSU/Improve)
[Part Collection](https://2018.igem.org/Team%3AFSU/Part_Collection)
[Parts](https://2018.igem.org/Team%3AFSU/Parts)

Attributions

[Team & Attributions](https://2018.igem.org/Team%3AFSU/Attributions)
[Collaborations](https://2018.igem.org/Team%3AFSU/Collaborations)
[References](https://2018.igem.org/Team%3AFSU/References)

[Judging Form](https://igem.org/2018_Judging_Form?team=FSU)
☰

# Experiments

## Plasmid Construction

![](https://static.igem.org/mediawiki/2018/8/8b/T--FSU--HiFi_Assembly.png)

Each plasmid was constructed using two gBlocks to form the insert and a chloramphenicol backbone. The first gBlock contains a biobrick prefix, the putative promotor, and the first 25 base pairs of the mRFP1 gene sequence. The second gBlock contains the full mRFP1 gene sequence, the BBa\_B0015 Double Terminator, and the biobrick suffix. Using the New England Biolab (NEB) Hifi DNA Assembly Master Mix protocols, the biobrick prefix and suffix allowed one end of both gBlocks to anneal to their respective sides of the Chloramphenicol backbone and the mRFP1 gene to anneal the two gBlock segments together. This process finalized the construction of our test devices and were then transformed into NEB 5-alpha cells for testing.

In order to test the activation response due to exposure to sound waves, a plasmid containing a putative promotor along with the red fluorescent protein sequence mRFP1 was first assembled for each sequence. This was achieved using the New England Biolab (NEB) Hifi DNA Assembly Master Mix; following these protocols, the plasmid constructed was believed to contain a chloramphenicol resistance gene backbone and an insert with the putative promotor sequence, a ribosome binding site, the mRFP1 gene, and a terminator. Each plasmid was then transformed into NEB 5-alpha, a strain of E. coli known for its transformation efficiency, and incubated on chloramphenicol plates to determine if the cells showed antibiotic resistance; this was our first indication if the plasmids were successfully transformed. Finally, the plasmids were purified from the growing cultures to verify the assembled plasmid sequences; there have been complications with the sequencing, but the plasmids are continuing to be analyzed.
The experiment was run on a 96-well plate with triplicates of each test cell with 2 different dilutions for a total of 6 wells for each test cell. Three wells contained test cells at the stationary phase and the other three wells contained cells diluted from the stationary phase by a factor of 10. A speaker was held on a stand 1 inch above the plate playing the desired frequency and left in an incubator for six hours to expose the cells to sound during the growth period. After the incubation, plates were read using the Synergy H1 Microplate reader from Biotek. The excitation-emission wavelengths for mRFP are 584 and 607nm but we decided to broaden the wavelength to 575 and 610nm in order to reduce wavelength interference. The plate reader allowed us to look at the Fluorescence level and Optical density. We Use a ratio of Relative Fluorescence Units to Optical density in order to assess the level of fluorescence of the cells. It was hypothesized that through our plasmid construction and testing, those putative promotors that were activated by sound would express mRFP1 and indicate higher readings of fluorescence compared to a control group of cells that were incubated with no sound. A PDF of all our protocols will be added to the during the wiki thaw.

[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:FSU/Results

[Home](https://2018.igem.org/Team%3AFSU)
[Description](https://2018.igem.org/Team%3AFSU/Description)
[Human Practices](https://2018.igem.org/Team%3AFSU/Human_Practices)
[Design](https://2018.igem.org/Team%3AFSU/Design)

Build & Test

[Experiments](https://2018.igem.org/Team%3AFSU/Experiments)
[Results](https://2018.igem.org/Team%3AFSU/Results)
[Improve](https://2018.igem.org/Team%3AFSU/Improve)
[Part Collection](https://2018.igem.org/Team%3AFSU/Part_Collection)
[Parts](https://2018.igem.org/Team%3AFSU/Parts)

Attributions

[Team & Attributions](https://2018.igem.org/Team%3AFSU/Attributions)
[Collaborations](https://2018.igem.org/Team%3AFSU/Collaborations)
[References](https://2018.igem.org/Team%3AFSU/References)

[Judging Form](https://igem.org/2018_Judging_Form?team=FSU)
☰

# 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.

![](https://static.igem.org/mediawiki/2018/7/7b/T--FSU--berkeley-fsu-promotors.png)

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.

![](https://static.igem.org/mediawiki/2018/0/01/T--FSU--berkeley-ambient-sound.png)

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

![](https://static.igem.org/mediawiki/2018/3/38/T--FSU--Berkeley-FSU-Promoters.Final.png)

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.

![](https://static.igem.org/mediawiki/2018/7/72/T--FSU--BIOFAB-Promoters-Performance.png)

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

![](https://static.igem.org/mediawiki/parts/1/1e/2_kHz_100_dB_comparative_graph_data_table.png)

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

![](https://static.igem.org/mediawiki/parts/0/04/8kHz_80_dB_data_table.png)

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

![](https://static.igem.org/mediawiki/parts/7/71/8_kHz_100_dB_sound_data_table.png)

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

![](https://static.igem.org/mediawiki/parts/1/1a/2_kHz_100_dB_data_table.png)

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