iGEM 2024 — Team rochester

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## Sidebar

[June](#JuneMonth)

[July](#JulyMonth)

[August](#AugustMonth)

[September](#SeptemberMonth)

[Wet Lab Protocols](#WetLabProtocols)

## June

#### June 17th

[Week of 6.17.24 Notebook Entries](https://static.igem.wiki/teams/5052/notebook/6-17-entriescompressed.pdf)

#### June 24th

[Week of 6.24.24 Notebook Entries](https://static.igem.wiki/teams/5052/notebook/6-24-entriescompressed.pdf)

---

## July

#### July 1st

[Week of 7.1.24 Notebook Entries](https://static.igem.wiki/teams/5052/notebook/7-1-entries.pdf)

#### July 8th

[Week of 7.8.24 Notebook Entries](https://static.igem.wiki/teams/5052/notebook/7-8-entries.pdf)

#### July 15th

[Week of 7.15.24 Notebook Entries](https://static.igem.wiki/teams/5052/notebook/7-15-entries.pdf)

#### July 22nd

[Week of 7.22.24 Notebook Entries](https://static.igem.wiki/teams/5052/notebook/7-22-entries.pdf)

#### July 29th

[Week of 7.29.24 Cloning Entries](https://static.igem.wiki/teams/5052/notebook/7-29-cloning-entries.pdf)
[Week of 7.29.24 CO2 Entries](https://static.igem.wiki/teams/5052/notebook/7-29-co2-entries.pdf)
[Week of 7.29.24 Cyanobacteria Entries](https://static.igem.wiki/teams/5052/notebook/7-29-cyanobacteria-entries.pdf)
[Week of 7.29.24 Notebook Entries](https://static.igem.wiki/teams/5052/notebook/7-29-entriescompressed.pdf)

---

## August

#### August 5th

[Week of 8.5.24 Notebook Entries](https://static.igem.wiki/teams/5052/notebook/8-5-entriescompressed.pdf)
[Week of 8.5.24 Promoter Entries](https://static.igem.wiki/teams/5052/notebook/8-5-promoter-entries.pdf)
[Week of 8.5.24 CO2 Entries](https://static.igem.wiki/teams/5052/notebook/8-5-co2-entries.pdf)
[Week of 8.5.24 Cloning Entries](https://static.igem.wiki/teams/5052/notebook/8-5-cloning-entries.pdf)

#### August 12th

[Week of 8.12.24 Notebook Entries](https://static.igem.wiki/teams/5052/notebook/8-12-entries.pdf)

#### August 19th

[Week of 8.19.24 Notebook Entries](https://static.igem.wiki/teams/5052/notebook/8-19-main-entries.pdf)
[Week of 8.19.24 Ethanol Entries](https://static.igem.wiki/teams/5052/notebook/8-19-etoh-entries.pdf)
[Week of 8.19.24 Cloning Entries](https://static.igem.wiki/teams/5052/notebook/8-19-cloning-entries.pdf)
[Week of 8.19.24 Promoter Entries](https://static.igem.wiki/teams/5052/notebook/8-19-promoter-entries.pdf)

#### August 26th

[Week of 8.26.24 Notebook Entries](https://static.igem.wiki/teams/5052/notebook/8-26-main-entries.pdf)

---

## September

#### September 2nd

[Week of 9.2.24 Notebook Entries](https://static.igem.wiki/teams/5052/notebook/9-2-entries.pdf)

#### September 9th

[Week of 9.9.24 Notebook Entries](https://static.igem.wiki/teams/5052/notebook/9-9-entries-1.pdf)

#### September 16th

[Week of 9.16.24 Notebook Entries](https://static.igem.wiki/teams/5052/notebook/9-16-entries-1.pdf)

#### September 23rd

[Week of 9.23.24 Notebook Entires](https://static.igem.wiki/teams/5052/notebook/9-23-entries-1.pdf)

---

## Wet Lab Protocols

[Wet Lab Protocols](https://static.igem.wiki/teams/5052/notebook/wet-lab-protocols.pdf)

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## Sidebar

[Design](#Design)

[Build](#Build)

[Test](#Test)

[Learn](#Learn)

## Our Process

---

### Design, Build, Test, Learn Cycle

In the design process of our biophotovoltaic cells, several prototypes were created to work towards maximizing the amount of voltage we were able to measure easily.

#### First Prototype:

Our first attempt at creating a functional biophotovoltaic cell was a very inspiring experience. This prototype was created using the components we had spent time testing and optimizing including our conductive ink, biological ink, and hydrogels soaked in 1% salt BG-11 media, as well as materials we already had in the lab including copper wire, tape, a multimeter, paper, and a pipette tip box to help maintain sterility. The protocol we followed was created by us and based on the design found in the published Sawa et al journal. Here, three layers of the conductive ink were printed using the screen printing technique while three layers of the biological ink were pipetted on top of the anode. This was because the concentration of CMC in our biological ink was too low, and the ink was not viscous enough to also be screen printed without leaking outside of the pattern. Each layer of ink was given time to dry before the next was printed to achieve a layering effect. Once the final layer of biological ink was dry, squares of hydrogels were placed on top of each anode and cathode pairing to facilitate a hydrogen gradient in our biophotovoltaic cells. Here, the hydrogels were sized so that they would be touching each other. Finally, two small lengths of copper wire were cut, stripped, and one was taped to the first anode while the second was taped to the last cathode in order to read the voltage across five of our cells. Our GW Instek GDM-8245 multimeter was used to probe the wires to show the voltage drop across our cells. Because the wires were connected to the conductive ink using a small piece of tape, only a small portion of the copper wire was making contact with the ink. Along with this, the connection was not very strong and the wires would often shift, especially when the probe would make contact with the wires, leading to very unstable readings. With these connection considerations in mind, the output of this first prototype was reading between 0.12-0.25 mV. To ensure the voltage being read was because of the cyanobacteria cells, we measured across a control setup that consisted of just the conductive ink without any cyanobacteria cells. When we measured this voltage it came back as zero volts, showing that the voltage we were measuring in our prototype was directly caused by the cyanobacteria. Another note is that we were not seeing an increase in voltage drop across biophotovoltaic cells in series. One reason for this could have been due to the cyanobacteria spreading from the anode to the cathode, creating one large anode across the paper and removing the flow of electrons that would be seen in a concentration cell. This spreading of the cyanobacteria was caused by the biological not having a high enough concentration of CMC, and not being viscous enough to remain strictly on the anode. A future improvement for subsequent prototypes is creating a more viscous biological ink with a CMC concentration of 8%.

![prototype1.1](https://static.igem.wiki/teams/5052/hardware/firstprototype1.png)
![prototype1.2](https://static.igem.wiki/teams/5052/hardware/firstprototype2.png)

Image 1: Two students measuring the voltage of Prototype 1

Image 2: Setup of Prototype 1

#### Second Prototype

After learning about the shortcomings of measurement of the first design, more supplies were purchased because it was clear that we needed to better the connections of the wires to the electrodes to obtain more consistent readings. The first change in materials included hydrogels that were cut down smaller to only cover the area of the anode and cathode of one biophotovoltaic cell at a time. Our first prototype was fluctuating between both positive and negative voltages, showing that the electrons were not strictly flowing from the anode to the cathode which we believed was caused by all of the hydrogels being in contact with one another. Along with this the hydrogels were soaked in a higher salt concentration of BG-11 media at 2% salt to encourage a stronger hydrogen gradient. Copper tape was used in place of the copper wires due to its high conductivity, flexibility, and increased contact surface area with the electrodes. By increasing the contact surface area a more stable reading was able to be measured. A new 87V Max multimeter was donated to our lab from Fluke and used in place of our old multimeter. With these changes in our new prototype, the multimeter was reading a consistent 17 mV across one biophotovoltaic cell, but also gave the same reading of 17 mV across four biophotovoltaic cells connected in series. In this prototype, we still were not able to measure an increase in voltage drop across multiple biophotovoltaic cells in series.

![prototype2](https://static.igem.wiki/teams/5052/hardware/secondprototype1.png)

Image 3: Setup of Prototype 2

#### Third Prototype

For the third prototype, the goal was to be able to measure an additive voltage drop in voltaic cells in series. To do this, one consideration we had was printing the adjacent anodes and cathodes of different voltaic cells closer together so the oxygen diffusion from the anode to the cathode is across a shorter distance. Having a shorter distance is both faster for oxygen diffusion because the molecules do not have to travel as far, and more favorable for the oxygen molecules compared to having to diffuse across a longer distance so they would diffuse at a greater rate. When the oxygen molecules reach the cathode, they participate in the reduction half-reaction that contributes to the formation of an electrical current, so allowing more oxygen molecules to travel to the cathode results in a greater contribution to the electrical current formation. Another observation we had in our second prototype was that when we measured the voltage across the cells using the multimeter, the voltage would begin to drop. To address this, we attached a 10 microfarad capacitor across four of the voltaic cells in series. Capacitors act to temporarily store built up voltage in a circuit, and release this energy when needed. With this addition we were able to measure 454 millivolts across the four cells, compared to 174.5 millivolts across the four cells without a capacitor. Measuring across one set of electrodes with the capacitor resulted in 92 millivolts, therefore we concluded that the voltage drop from the four cells in series was additive, and our improvements worked.

![prototype3.1](https://static.igem.wiki/teams/5052/hardware/thirdprototype1.png)
![prototype3.2](https://static.igem.wiki/teams/5052/hardware/thirdprototype2.png)

Image 4: Capacitor connected across single pair of electrodes in Prototype 3

Capacitor connected across four pairs of electrodes in Prototype 3

![”Prototype-voltage”](”https://static.igem.wiki/teams/5052/hardware/prototype-1.png”)

Figure 23: Graph of the average voltage measured from each prototype

#### Future Directions

For subsequent prototypes, one goal is to be able to light up a diode. Having proven that we were able to build up a voltage across multiple biophotovoltaic cells in our third prototype, the next step would be to print more biophotovoltaic cells in series and measure if their voltage drop would continue to be additive. One consideration for this setup is that the more biophotovoltaic cells that are printed, the larger the value of the resistance will be between the first and last cells. Because of this, naturally, some voltage output will be lost due to the resistance. To combat this another design we could test is connecting multiple series of biophotovoltaic cells in parallel. There is no voltage drop over cells in parallel, but the distance between them will decrease which will decrease the resistance experienced by the current. Another way we would be able to build up more voltage would be by adding more capacitors to the individual biophotovoltaic cells connected in series. This would not only allow the biophotovoltaic cells to charge up their voltage before releasing it but would also give a more stable reading to the multimeter when measuring a voltage across the circuit.

Another future direction would be testing these biophotovoltaic cell prototypes using transformed cyanobacteria. Currently, we have only tested these prototypes using wild-type cyanobacteria, *Synechocystis* sp. PCC 6803. A part of our wet lab team’s efforts was put into genetically modifying cyanobacteria with psbE and psbF. The alpha and beta subunits respectively of cytochrome b559 play a role in the generation of electrons from water on photosystem II 1. Transforming cyanobacteria with these has been proven by the wet lab team to work, so the next step would be growing enough transformed cyanobacteria to test our biophotovoltaic cells. The assumption would be that these transformed cells would be able to produce more voltage than the wild-type strain.

A third future direction would be looking into incorporating ethanol distillation into our prototypes. The ethanol production by our cyanobacteria is one of our modules in the wet lab aspect of our project. With this, we have found that ethanol can be distilled from the hydrogels sitting on top of the cyanobacteria cells in our biophotovoltaic cell design. This distillation process creates a cleaner way to produce ethanol and would be a strong addition to an upscaled version of our biophotovoltaic cell systems.

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## **Characterization**

Our wet lab team worked to characterize parts that were successfully cloned. This includes carbonic anhydrase from N. vulgaris and pdc and adhB from Z. mobilis.

Carbonic anhydrase from *N.vulgaris* is known to be heat-resistant2. To test if our heat-resistant carbonic anhydrase would allow *Synechocystis* sp PCC 6803 to sequester a similar amount of CO2 compared to wildtype under room conditions, we used an airtight Tupperware container to test the decreasing concentration of CO2 over time. Unfortunately, the Tupperware we used wasn’t airtight even after we parafilmed the container. Therefore, we decided to take the hourly measure of CO2 concentration in an empty container. This would be our control to see if cyanobacteria would affect how fast the CO2 concentration dropped. Thus, we can assume the additional decrease in concentration would be from the cyanobacteria absorbing the CO2.

Pdc and adhB from *Z.mobilis* are known for their high efficiency. Pdc catalyzes the conversion of pyruvate to acetaldehyde. Acetaldehyde then is converted to ethanol by adhB. To test if PDC and adhB would allow *Synechocystis* sp PCC 6803 to produce ethanol we set up a titration curve using Schiff’s reagent. Schiff’s reagent is often used for ethanol detection in biological staining. When Schiff’s reagent reacts with ethanol it turns the sample a bright magenta color. We used this change of color to create a standard curve and to detect and quantify the ethanol produced by BL21 *E.coli* strain transformed with pdc and adhB.

We step up two controls of parafilm Tupperware with a CO2 monitor to test how fast the air leaves the box with no cyanobacteria. We also set up a box with wildtype *Synechocystis* sp. PCC 6803 cyanobacteria was taken from a seed culture originally inoculated from a colony on a BG-11 agar plate and grown at both 30°C with constant rotation at 200 rpm. The O.D.730 for the sample was 0.337. Our modified sample to test was S*Synechocystis* sp. PCC 6803 with our modified CA, “coordinated active site domain” of the enzyme carbonic anhydrase that promotes carbon sequestration. The O.D.730 for the sample was 0.400. All the samples were placed in identical Tupperware containers and parafilm in the same way with parafilm along the edge of the container. They were placed in a CO2 incubator brought up to 1%. Once the samples reached 1% CO2, we took them out of the incubator and placed them in room air still sealed. We waited up to 11 hours to see the drop in concentration of CO2.

After testing both the wildtype *Synechocystis* sp. PCC 6803 and our modified *Synechocystis* strain with the modified carbonic anhydrase from N. vulgaris (BBa\_K5052911), we found that the wildtype cyanobacteria decreased carbon levels at a faster rate than the modified strain. The wildtype had a carbon reduction of -184 ppm/hour, while the modified strain's was -92.7 ppm/hour.

These results conclude that BBa\_K5052911 does effect the carbon sequestration at room temperature since the rate of carbon capture was slower than wildtype.

EtOH Detection Experiments
Schiff’s Reagent Indicator Plates

Schiff’s reagent is a chemical solution that can be used to detect the presence of aldehydes, primary, and secondary alcohols. It is created by decolorizing a dye, typically fuchsin, with sulfur dioxide. When an aldehyde is present, the reagent restores the dye’s original magenta color, making it a useful test in biological staining1. Using the protocol by Jason Dexter and Pengcheng Fu, we attempted to make Schiff’s reagent indicator plates which could be used to test ethanol production in transformed BL21 strain and Synechocystis sp. PCC68032. Following the protocol, we attempted to make LB-agar indicator plates with 2% Schiff’s reagent. These plates would be used to test BL21 E.coli strain and Synechocystis sp. PCC 6803 was transformed with our ethanol cassette for ethanol production. A change in the color of the plates would indicate ethanol production2.
First, to confirm that Schiff’s reagent can detect alcohol we added 1% Schiff’s reagent to solutions of 100% ethanol, 70% ethanol, 70% isopropanol, and H2O (Figure 1). Like ethanol, isopropanol is an alcohol. Unlike ethanol, it is a secondary alcohol. Observing the intensity of magenta would also allow us to observe how much it reacts to different concentrations of ethanol as well as how differently it reacts between primary and secondary alcohols at the same concentration. The Schiff’s reagent turned all alcohol samples bright magenta confirming that it can be used to detect both primary and secondary alcohols in solution. Qualitatively, 100% ethanol showed the deepest shade of magenta. Between 70% ethanol and 70% isopropanol, it appeared that Schiff’s reagent reacts more with primary alcohols than secondary.
Figure 1: From left to right: tube with 100% ethanol, 70% ethanol, 70% isopropanol, H20
After demonstrating that our team was able to use Schiff's reagent to detect alcohols like ethanol, we attempted to make indicator plates by adding 4 mL of Schiff’s reagent to 200 mL LB-agar-kan (2% Schiff’s) to see if our colonies were producing ethanol. Upon adding Schiff’s reagent, the solution turned a bright red color (Figure 2). This indicated that Schiff’s reagent either reacted to something in LB-agar-kan or the LB-agar-kan was too hot which could affect the reactivity of Schiff’s reagent.
Figure 2: LB-agar-kan turned bright red after the addition of Schiff’s reagent.
We then attempted to overlay 200 uL of Schiff’s reagent onto premade LB-agar plates to see if Schiff’s reagent was going to react with cooled LB-agar. The plates were stored in the refrigerator overnight to make sure they were cooled enough. The LB-agar plates turned light pink upon adding the reagent, indicating a reaction with the LB media itself or with the agar (Figure 3).
Figure 3: The plate on the left had Schiff’s reagent added to it; the plate in the middle had Schiff’s reagent overlaid on it; the plate on the right is an LB-agar plate.
Following these results, we tested liquid LB media by adding 20 uL Schiff's to 1 mL LB to see if the Schiff’s reagent was reacting specifically with the agar or with LB media. LB turned a slight pink color. We then added 20 uL of 70% EtOH, but no visible difference was observed. After that, we tried adding 80 uL more of 70% EtOH to the same sample to get 100 uL of the final volume of Schiff’s reagent, but still, no visible difference was observed. Finally, we tried adding 80 uL more of Schiff's reagent for the final volume of 180 uL but still, no color difference was observed (Figure 4). These results led us to the conclusion that Schiff's reagent would not be a reliable metric for detecting ethanol in LB media. Considering that Schiff’s reagent was reacting to something in LB media, we decided to use the M9 minimal medium instead due to its ability to support cellular growth and the lack of components such as yeast extract and tryptone that are present in LB media.
Figure 4: The tube on the left contained LB+EtOH with Schiff’s; the tube in the middle contained LB with Schiff’s; the tube on the right contained LB.
To see if we can use Schiff’s reagent to detect and quantify our ethanol production in M9, a titration curve was produced. Schiff’s reagent was cooled in the fridge. Samples were vortexed after adding Schiff’s and let sit for an hour before reading the plate on the plate reader at 462 nm (K. Tennakone et al., 1987). The reading was performed again after 2 hrs and the curve was generated (Figure 5). The produced titration curve is linear which indicates a linear association between the percentage of ethanol and absorbance.
1mL of M9 + 100mL Schiff's (10%)
70% ethanol
50% ethanol
30% ethanol
20% ethanol
10% ethanol
M9 with Schiff's
M9

![Liquid cyanobacteria at different O.D.s compared to Kimwipe box](https://static.igem.wiki/teams/5052/experiments/wet-1.png)
Figure 5: The titration curve
After performing the titration curve using the M9 medium, we saw that the reagent would be a reliable metric for ethanol detection because it showed increasing intensity of magenta with increasing concentrations of ethanol. Knowing this, we tested our E. coli transformed with the complete ethanol cassette to see if our parts worked. If our ethanol cassette is functional, we would be able to observe a difference in the intensity of magenta over time. BL21 + EtOH was grown at 37℃ in M9 minimal medium shaking at 100rpm. Since the ethanol cassette is under the control of the dark inducible promoter, PompC, the culture was covered with tin foil to limit the amount of light reaching the culture to allow the expression of AdhB and Pdc. The next day, we aliquoted 1 mL of just M9 medium as our negative control, BL21 strain with EtOH plasmid, and DH5a strain with the empty vector, pAM4891. We then added 100uL of Schiff's reagent and observed a color change (Figure 6).
Figure 6: From left to right: M9 + Schiff’s, DH5a + pAM54891 + Schiff’s, BL21 + EtOH + Schiff’s
The sample BL21 + EtOH + Schiff’s shows a significantly deeper shade of magenta than DH5a + pAM54891+ Schiff’s and M9 + Schiff’s alone. These results hint at the presence of ethanol as well as the functionality of AdhB and Pdc biobricks.
Our next goal was to quantify the ethanol produced by our transformed E. coli strains. This was done by setting up a 96-well plate similar to how we set up our titration curve.
![Liquid cyanobacteria at different O.D.s compared to Kimwipe box](https://static.igem.wiki/teams/5052/experiments/wet-2.png)
Figure 7: The results of quantification

The results of a plate reader at A462 were promising, the difference between pAM4892 and the EtOH biobricks transformed E.coli was visible, but the data also indicated that the sample with E.coli transformed with pAM4891 contained 14% ethanol and the difference between the absorptions was not significant. This indicated a reaction of Schiff’s reagent with the components of M9 media.
Following these results, we decided to perform distillation. This served two purposes. Mainly to remove media that was affecting our results. The other purpose of performing distillation was to consider our overall design. The biophotovolteic cells were designed to be covered by hydrogel, so the ethanol secreted by cyanobacteria would go to the hydrogel, which would need to be distilled to extract the ethanol.
To see if our method for the distillation of ethanol from the media would work we had hydrogels soak up the media and ethanol produced by our E.coli, remove it from the culture, and heat it to distill the ethanol from the hydrogel. To achieve this we made 70% ethanol and 50% ethanol solutions with a total volume of 6 mL for each sample. We set aside 1 mL of each sample as our control and tested them with Schiff’s reagent to compare the color of the sample to the results we got out of the hydrogels. We let hydrogels soak for an hour in the samples. After the soaking, the hydrogel was put on a glass petri dish, covered with a 60 mm petri dish, and heated up to 78℃ until the droplets of liquid started to appear on the top of the petri dish. The 10 uL of Schiff’s reagent was added to the formed liquid and results were recorded.
Figure 8: Results after adding 10 uL of Schiff’s reagent.
The results showed that both control ethanol samples and ones distilled from hydrogen both turned the same shade of magenta indicating the presence of ethanol and proving that our distillation method worked (Figure 8). After getting the results, we tested just hydrogels. We soaked one hydrogel in the BG-11 media and another in water as our control. We used the same setup with the Petri dishes, unfortunately, Schiff's reagent reacted with the plate. After leaving it for a while all plates turned magenta. Following these results, we attempted distillation with a different collection vessel - a beaker. We soaked hydrogel in 50% EtOH, 70% EtOH, water and tested the beakers by adding 100 uL Schiff's and letting them sit for 10 min. The idea was to precoat the beakers in Schiff's which would allow us to see if there's ethanol in real time accurately. The beaker was also causing the Schiff's reagent to react but only slightly. Distillation was run for five minutes at 80℃ and 10 uL of Schiff's was added to the samples. The color change was observed.
Figure 9: Left to right: water-soaked hydrogel distillation, 50% ethanol-soaked hydrogel distillation, 70% ethanol-soaked hydrogel distillation.
The intensity of the magenta color was roughly the same between the two ethanol distillations, indicating the same concentration of ethanol distilled (Figure 9). These results proved that our distillation method. can be used to extract ethanol from hydrogels, so our next step was to perform distillation on the supernatants from our DH5a E. coli strain transformed with our ethanol plasmid and BL21 E. coli strain transformed with our ethanol plasmid to see if they produce ethanol. Four samples were set up to perform the distillation. Two were grown on the plates and two were grown in the liquid media. In both cases, E.coli were grown in the dark at 37℃. The plate samples were grown on LB-agar-km plates and liquid samples were grown in LB-km 5 mL cultures overnight. For plate samples, hydrogels were laid over the top of the lawn of E.coli. For liquid samples, hydrogels were soaked in the media extracted from liquid cultures after spinning them down and removing the pellet for 1hr. For the control, we soaked hydrogel in LB-km alone for 1hr. All samples were distilled and captured in 50 mL beakers. A 100 uL of each sample was collected in 1.5mL tubes and 10 uL of Schiff's reagent was added.
Figure 10: Results after 1 min after adding 10% Schiff’s. Left to right: DH5a + EtOH on solid, BL21 + EtOH on solid, DH5a + EtOH in liquid, BL21 + EtOH in liquid, LB.
The absence of color change in the LB sample indicated that distillation removed all possible components that could react with Schiff’s reagent, which means that the color change in other samples could be only due to the presence of ethanol. The BL21 sample on solid media showed a much darker shade of magenta than the DH5a sample on solid media, which makes sense considering that BL21 is an expression strain of E.coli. These results once again proved that our AdhB and Pdc biobricks work in E.coli (Figure 10).
To quantify these results Schiff’s reagent ImageJ titration curve was performed based on Figure 9. The following results were produced:
![Liquid cyanobacteria at different O.D.s compared to Kimwipe box](https://static.igem.wiki/teams/5052/experiments/wet-3.png)
Figure 11: Schiff’s reagent ImageJ Titration Curve
![2](https://static.igem.wiki/teams/5052/hardware/aa2.png)
Figure 12: Graph of ethanol concentration
Using the data from ImageJ Figure 10 was produced. The BL21 strain transformed with our EtOH biobrick and grown on solid media showed 75.02% ethanol per volume. The BL21 strain transformed with our EtOH biobrick and grown in liquid media showed 22.25% of ethanol per volume. Both results are significantly larger compared to the LB sample with 1.95% ethanol (Figure 11, 12).

## **Cyanobacteria**

Our team utilized two main cyanobacterial strains: *Synechocystis* sp. PCC 6803 and *Synechococcus* sp. UTEX 3154, in order to power our biophotovoltaic fuel cell. Throughout this process, we learned and documented methods for their cultivation by conducting several experiments and setting up our own assays to learn the best conditions for optimal cyanobacteria growth.

[Media Assay](#MediaAssayDD)

When reviewing existing research on cyanobacteria, we found that two types of media, BG-11 and Artificial Seawater, are commonly used for growing marine species such as *Synechococcus* sp. UTEX 3154. To determine which medium would support greater growth yield, we decided to compare the effectiveness of BG-11 and Artificial Seawater.

For our experiment, we used 1 mL samples of both BG-11 and Artificial Seawater inoculated with *Synechococcus* sp. UTEX 3154. Both samples were kept in a 38°C shaking incubator at 100 rpm with a 25% light intensity setting on our LED grow lights.

![Graph 1](https://static.igem.wiki/teams/5052/experiments/wlgraph1.png)

In the first 24 hours, Artificial Seawater appeared to promote better growth, with an O.D. (optical density) of 0.018 compared to BG-11’s 0.005. However, by day 5, BG-11 surpassed Artificial Seawater, reaching an O.D. of 0.033, while Artificial Seawater measured at only 0.022. This trend continued as the experiment progressed. After 11 days of growth, BG-11 showed exponential growth whereas Artificial Seawater plateaued.

Although Artificial Seawater initially promoted faster growth, BG-11 sustained a higher growth rate over time. Based on these results, we concluded that BG-11 is the more effective medium for cultivating cyanobacteria in this experiment, and we continued to use it as our primary growth medium.

[Bicarbonate Assay](#BicarbonateAssayDD)

In several studies on cyanobacteria growth, we observed that bicarbonate was added as an additional carbon source to support cyanobacterial growth1. To determine its potential benefits for our system, we conducted our own tests to evaluate its effectiveness.
 To assess the potential advantages and disadvantages of bicarbonate flashing, the process of regularly adding bicarb to stimulate growth, we conducted several tests using 1 mL samples of *Synechococcus* sp. UTEX 3154 in different media. Samples were prepared by taking inoculant from the seed culture purchased from UTEX. We compared the effects of adding bicarbonate daily versus every other day in both BG-11 and Artificial Seawater (Artificial Seawater). Both samples were given an initial 24-hour growth periO.D. with no bicarbonate, and were kept in a 38°C shaking incubator at 100 rpm with a 25% light intensity setting on our LED grow lights.

![BicarbonateAssay](https://static.igem.wiki/teams/5052/hardware/addbicarcassay.png)

By Day 4, all samples showed relatively similar O.D.s, ranging from 0.016 to 0.027 across the five test groups. However, significant changes emerged by Day 6. BG-11 liquid media with daily bicarbonate addition nearly doubled overnight, increasing from an O.D. of 0.065 to 0.102, while BG-11 liquid media with bicarbonate added every other day only increased from 0.040 to 0.057. In contrast, Artificial Seawater with bicarbonate every other day showed a decline from 0.048 to 0.026, consistent with our earlier findings that Artificial Seawater is less effective at sustaining cyanobacterial growth. Artificial Seawater with daily bicarbonate addition exhibited minimal growth, with an O.D. of just 0.006.

By Day 11, this trend persisted. The sample with the highest O.D. was BG-11 liquid media with daily bicarbonate addition at 0.347, followed by BG-11 with bicarbonate every other day at 0.285. The control sample, BG-11 with no bicarbonate added, had an O.D. of 0.109. Both Artificial Seawater samples, with and without bicarbonate, ended with O.D.s under 0.01, confirming that Artificial Seawater is not as effective for supporting sustained cyanobacterial growth.

While the samples that were flashed with bicarbonate were able to grow at a higher rate than those that were not, we observed that the bicarbonate was forming flakes in our media and was preventing our cyanobacteria from fully shaking in our incubator. Therefore, bicarbonate flashing was a useful way to grow up a culture with a very small starting O.D., but it was not useful when our samples reached higher O.D.s as sufficient gas-exchange was not efficient with the excess bicarbonate in the culture.

[pH Assay](#pHAssayDD)

As *Synechococcus* sp. UTEX 3154 is an uncharacterized species, we wanted to investigate whether it would thrive better in more alkaline conditions, as marine environments tend to be slightly basic2>. We tested two different pH levels: 7 (the natural state of BG-11) and 8 (to simulate more basic, marine-like conditions).

In order to conduct this assay, we set up two cultures: one with *Synechococcus* sp. UTEX 3154 in BG-11 1% NaCl liquid media with at pH 7 (no additional base), and another in BG-11 1% NaCl liquid media adjusted to pH 8 using KOH and Tris HCl buffer. Both samples were kept in a 38°C shaking incubator at 100 rpm with a 25% light intensity setting on our LED grow lights.

![Graph 1](https://static.igem.wiki/teams/5052/experiments/wlgraph2.png)

Over 11 days of growth, the results were clear. The culture at pH 7 reached an O.D. of 0.127, showing consistent growth throughout the experiment. In contrast, the pH 8 culture only reached an O.D. of 0.006 and never entered the log growth phase. Based on these results, we concluded that UTEX 3154 grows best at pH 7 and decided to maintain the natural pH of BG-11 without further adjustment.

[Salt Assay](#SaltAssayDD)

Since we were working with *Synechococcus* sp. UTEX 3154, a marine cyanobacterial species, we wanted to determine if salt concentration played a significant role in its growth.

 We tested this with four different salt concentrations: 0% (no salt), 1%, 1.5%, and 2%. Four test tubes contained 2 mL of BG-11 media each with their varying levels of salt concentration and were inoculated with *Synechococcus* sp. UTEX 3154. Both samples were kept in a 38°C shaking incubator at 100 rpm with a 25% light intensity setting on our LED grow lights.

 ![Graph 1](https://static.igem.wiki/teams/5052/experiments/wlgraph3.png)

After 11 days of growth, the sample with 1% salt concentration had the highest O.D., followed by 0% salt, then 1.5%, and finally 2%, which had only reached an O.D. of 0.02.

To further investigate, we extended the assay until day 28 to observe the long-term trends. After several weeks, the sample with 0% salt (the control) showed the highest O.D., revealing it was the most effective for growth. This assay allowed us to determine that no added salt in the BG-11 media was the most optimal condition for growing our marine cyanobacterial species.

[Temperature Assay](#TemperatureAssayDD)

From our initial research, we believed that the optimal temperature for growing UTEX 3154 was 38°C with slight shaking, which is the protocol we followed for early experiments 3. However, after consulting with Dr. Alistar McCormick from the University of Edinburgh, a cyanobacteria expert, we learned that such a high temperature could lead to excessive media loss. Based on this advice, we decided to test our cyanobacteria cultures at 30°C and 38°C to determine the optimal temperature for growth.

We set up BG-11 liquid cultures inoculated with *Synechococcus* sp. UTEX 3154 at both 30°C and 38°C, maintaining a speed of 200 rpm and a 75% light intensity setting on our LED grow lights.

![Graph 1](https://static.igem.wiki/teams/5052/experiments/wlgraph4.png)

Over time, the *Synechococcus* sp. UTEX 3154 in 30°C had an exponential growth curve with a max O.D. of 1.146 after 16 days while the *Synechococcus* sp. UTEX 3154 in 38°C was only able to reach a max O.D. of 0.277 in 25 days. Based on these findings, we adjusted all future experiments to grow UTEX 3154 at 30°C for optimal growth.

[Antibiotic Assay](#AntibioticAssayDD)

In order to conduct successful homologous recombination to modify cyanobacterial genomes, a high concentration of antibiotics is needed to ensure that each copy of the chromosome is included. For example, when discussing with Dr. Toby Call from the University of Cambridge, we came across his PhD thesis regarding using cyanobacteria in biochemical systems. In his thesis, up to 500 ug/mL of kanamycin was used to isolate successful transformants 4. However, we decided to conduct our own assay to determine the optimal amount as the concentration of antibiotics needed when using a shuttle vector such as RSF1010 has not been extensively studied. We designed an assay to determine the maximum amount of antibiotic that our cyanobacterial strains could handle.

We began with 5 test tubes each with 2mL of Synechocystis sp. PCC 6803 and 5 test tubes with 2 mL of *Synechococcus* sp. UTEX 3154. Each culture of cyanobacteria started at an O.D. of 0.3 at a wavelength of 730 nm. 0 ug/mL, 25 ug/mL, 50 ug/mL, 100 ug/mL, or 200 ug/mL were added to each of the test tubes for each sample. The samples were kept with constant light and were shaken at 200 rpm. The O.D. at a wavelength of 730 nm was measured each day for a week of growth.

![Graph 1](https://static.igem.wiki/teams/5052/experiments/wlgraph5.png)
![Graph 1](https://static.igem.wiki/teams/5052/experiments/wlgraph6.png)

Our results showed that a minimum of 50 ug/mL was needed to successfully kill wild-type cyanobacteria While 25 ug/mL of kanamycin was sufficient in killing wild-type *Synechococcus* sp. UTEX 3154, we observed a dip followed by a sustained O.D., indicating that a spontaneous mutant may have arised. This assay allowed us to determine that 50 ug/mL of kanamycin is needed to successfully isolate transformed colonies.

## Cloning

We have identified seven parts that we hypothesized would improve the efficiency of our strains to sequester carbon, generate electricity, and produce ethanol. However, genetic manipulation of cyanobacteria is limited. Homologous recombination is the standard but has a lengthy selection process due to the need to ensure each copy of the chromosome in a cell contains the gene of interest, of which there could be hundreds1. The ploidy of cyanobacteria is also dependent on the growth stage which makes reliable modification via homologous recombination more difficult. To avoid these difficulties, our team opted to modify using a shuttle vector replicable in both *E. coli* and cyanobacteria, RSF1010. RSF1010-based plasmids are the only well-known shuttle vector between E. coli and cyanobacteria, consequently, we have developed novel solutions to ease the engineering process, including a promoter swap system as a way to efficiently switch out the promoter controlling our gene of interest, and an improved method of gene integration as a way to introduce two independent genes into the same cell without the risk of incompatibility of RSF1010-based plasmids. This allowed us to create cassettes of composite parts with all necessary components to address one of our three modules which we can modify our strains with.

## Promoter swap

This promoter swap system is based on traditional cut-and-paste cloning. Traditional cut-and-paste cloning was an attractive solution to us as the smallest of recognition sites can be easily added without disturbing the function of the full composite part. Two restriction sites are placed on either end of the promoter, DraIII cut site is placed upstream of the promoter and MluI cut site is in between the ribosome binding site and the start codon of the DNA coding sequence. Promoters can be exchanged via double digest with DraIII and MluI, performing a gel extraction of the vector, and inserting your new promoter of choice.

We have worked to modify three cyanobacterial promoters: Pcpc560–a constitutive promoter, PompC–a dark inducible promoter, and cLac145–an IPTG inducible promoter.

We characterized our promoter swap system and measured success by comparing EYFP expression in parts with promoters swapped to their native sequence. We took EYFP controlled by PompC (BBa\_K5052902) and cLac145 (BBa\_K5052900) and swapped out the promoters. For PompC + EYFP, we cut out PompC with DraIII and MluI to replace it with cLac145 and separately with Pcpc560. For cLac145 + EYFP, we cut out cLac145 with the same pair of restriction enzymes to replace it with PompC.

After performing traditional cut-and-paste to insert the respective promoters, the ligated products were used to transform DH5a E. coli. These transformants were grown on LB-agar-kan plates at 37C in the dark overnight. The next day, these plates were taken for fluorescent imaging.

![3D Polynomial Surface Model](https://static.igem.wiki/teams/5052/modeling/1.png)

This image was quantified for fluorescence on ImageJ by taking the area of the plate and calculating the intensity of the fluorescence. With this value, we can divide by the background values to find the corrected total cell fluorescence2.

![3D Polynomial Surface Model](https://static.igem.wiki/teams/5052/experiments/2.png)

When comparing swapped promoters to their native sequence, we see that the levels of fluorescence are vastly different. cLac145 + EYFP has a corrected total cell fluorescence of 11.93 compared to after we swapped it with PompC where the corrected total cell fluorescence increased to 16.321. When looking at PompC + EYFP, it shows a corrected total cell fluorescence of 18.875 which decreased greatly to 10.066 when swapped with Pcpc560 and to 11.354 when swapped with cLac145. Furthermore, comparing the strength of fluorescence between parts sharing the same promoter shows similar results, like between cLac145 + EYFP and PompC insert cLac145 which have a corrected total cell fluorescence of 11.93 and 11.354 respectively.

## Super-plasmid assembly

Plasmid incompatibility refers to the inability for two or more plasmids to coexist in one bacterial cell. This most often occurs due to both plasmids possessing the same replicon with specific controlling elements, or competition from similar partitioning elements1. To combat this issue, we developed a system of ‘super’ plasmids, as well as a plan to co-culture cyanobacteria such that each cell would only need to contain one plasmid of interest.

Each of the 3 modules of our project, carbon sequestration, electricity generation, and ethanol production had a planned super plasmid. This plasmid would contain all of the Biobricks that we planned to transform into cyanobacteria to get it to express the desired biological pathways. Additionally, the plasmid would contain the proper promoters for gene expression, and the kanamycin resistance genes necessary to test if our transformations were successful. In essence, each plasmid would be a self-contained system for the purposes of expressing multiple proteins of interest.

The carbon sequestration plasmid was planned to contain the SbtA and BicA genes, which both encode bicarbonate transport complexes. This plasmid being successfully incorporated into our cyanobacteria would result in a greatly increased uptake of carbon dioxide from the atmosphere.

![3D Polynomial Surface Model](https://static.igem.wiki/teams/5052/experiments/3.png)

Our electricity production plasmid was designed to carry the PsbE and PsbF genes that together express proteins for the cytochrome b559 protein complex. This complex is an essential part of photosystem 22, and should greatly increase the voltage produced by our cyanobacteria if properly formed.

![3D Polynomial Surface Model](https://static.igem.wiki/teams/5052/experiments/4.png)

As the final stage of our planned 3 modules, the ethanol plasmid carries the genes pdc and adhB from the *Z. mobilis* bacterium. These genes encode for the enzymes pyruvate decarboxylase and alcohol dehydrogenase respectively, which serve as critical components of the ethanol fermentation pathway. When expressed in cyanobacteria, the production of ethanol will be enabled, allowing us to collect a valuable byproduct from the cyanobacteria.

![3D Polynomial Surface Model](https://static.igem.wiki/teams/5052/experiments/5.png)

Creating the super plasmids in reality was conceptually very simple. Each biobrick had previously been ligated into the modified RSF1010-mobAY25F cloning vector through simple cut-and-paste. From there, based on the availability of restriction enzymes, one of the Biobricks would be cut out of its vector with a double digest, while the other vector would undergo a single digest to prepare it for ligation with the other gene of interest. Each super plasmid could be transformed into *E. coli* and grown in large amounts to prepare for eventual transformation into our cyanobacteria. However, this process was fraught with difficulty and setbacks.

Despite the simplicity of the planned experiments, we saw very little success with early attempts to digest our vectors and ligate both Biobricks together into one cloning vector. It is likely that the large plasmid resulting from having two genes present reduced the likelihood of proper ligations and transformations occurring. Most selection tests with bacteria indicated a failed transformation, and PCR analysis of the plasmids confirmed this negative result.

Ultimately it ended up being a matter of patience before we were able to confirm positive results in terms of a successful transformation. These results came about through a colony PCR analysis. Every time a transformation was performed, the resultant cells would be plated on a kanamycin selection plate. Any colonies that grew would be run through colony PCR using primers we designed specifically to multiply our genes of interest. When run against a positive control on an agarose gel, this approach enabled us to verify the presence of a successful super plasmid.

![3D Polynomial Surface Model](https://static.igem.wiki/teams/5052/experiments/6.png)
![3D Polynomial Surface Model](https://static.igem.wiki/teams/5052/experiments/7.png)

As seen in this comparison with an idealized version from SnapGene, the bands are exactly where we would expect them to be (accounting for different reactions being loaded into each well). This demonstrates that both the super electricity plasmid containing PsbE and PsbF, and the super ethanol plasmid containing pdc and adhB are present in separate *E. coli* cultures. Our carbon super plasmid was not able to be successfully ligated and transformed during the scope of this project.

After confirmation of the presence of our proper super plasmids, the colonies containing the successful result were grown quickly and allowed to multiply to a high degree. This was in preparation for the next step, natural transformation of cyanobacteria. The procedures and testing for this part of the project can be found in our Cyanobacteria section.

[1]Thomas, C. M. (2014) Plasmid incompatibility. Molecular Life Sciences 1–3.

[2]Pakrasi, H. B., Williams, J. G., and Arntzen, C. J. (1988) Targeted mutagenesis of the psbe and psbf genes blocks photosynthetic electron transport: Evidence for a functional role of cytochrome B559 in Photosystem ii. The EMBO Journal 7, 325–332.

## Sidebar

[Overview](#Overview)

[Carbon Sequestration Model](#Sequester)

[Background](#Sequester-Background)

[Goals](#Sequester-Goals)

[Assumptions](#Sequester-Assumptions)

[Approach](#Sequester-Approach)

[Model Simulation and Results](#Sequester-Results)

[Discussion](#Sequester-Discussion)

[Future Directions](#Sequester-Future)

[Electricity Model](#Electricity)

[Background](#Electricity-Background)

[Goals](#Electricity-Goals)

[Assumptions](#Electricity-Assumptions)

[Approach](#Electricity-Approach)

[Model Simulation and Results](#Electricity-Results)

[Discussion](#Electricity-Discussion)

[Future Directions](#Electricity-Future)

[Cyanobacterial Growth Model](#Grow)

[Background](#Grow-Background)

[Goals](#Grow-Goals)

[Assumptions](#Grow-Assumptions)

[Approach](#Grow-Approach)

[Model Simulation and Results](#Grow-Results)

[Discussion](#Grow-Discussion)

[Future Directions](#Grow-Future)

## **Overview**

We created several modeling projects to support the wet lab and the development of the hardware. The modeling projects focus on the most important parts of the project which include the ability of the cyanobacteria to sequester carbon dioxide from the environment, and the ability for the cyanobacteria to generate electricity.

The Main goals of our model:

1. To aid the hardware team in designing the most optimal pattern of electrodes for the biophotovoltaic cell to result in the maximum output of electricity.
2. To provide information to wet lab about the ability of the wild type and modified cyanobacteria to sequester carbon dioxide.

---

## **Carbon Sequestration Model**

### Background

### Goals

This model aims to provide more information about two questions which are explored in the Wet Lab:

1. How does the overexpression of SbtA and BicA in cyanobacteria affect the efficiency of carbon sequestration?
2. How does replacing the Wild Type Carbonic Anhydrase with N. vulgaris Carbonic Anhydrase in cyanobacteria affect the efficiency of carbon sequestration?

### Assumptions

### Approach

### Model Simulation and Results

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Asperiores molestiae fugit, necessitatibus totam et est atque, facilis quod eveniet sint, hic eius alias provident dolorum cupiditate deserunt! Quaerat ducimus tempora magni nesciunt iste unde rerum corrupti laboriosam odio accusamus. Asperiores sint, impedit, error perferendis voluptatum corporis voluptas necessitatibus, officia dolore sapiente laboriosam illum. Reiciendis at aliquid ut praesentium architecto perspiciatis sapiente recusandae sequi tempore odit doloribus error placeat, animi eum ducimus, voluptatum ipsam atque rerum accusantium laudantium sit explicabo eos. Placeat suscipit autem fugiat qui at, a expedita iste perferendis aperiam voluptatem et sed exercitationem ea maxime porro eius accusamus possimus nam esse recusandae quaerat tenetur? Eum eius quaerat temporibus adipisci saepe quos magnam necessitatibus est veritatis ipsa id esse earum cumque beatae, placeat dolore error blanditiis culpa excepturi nesciunt repellat amet! Unde in veritatis ipsam totam, repellat dolores iure soluta natus molestiae blanditiis eaque quasi suscipit, cupiditate magni? Numquam nostrum rem quos architecto hic, eveniet iusto suscipit delectus aspernatur voluptatem animi aliquam eligendi, inventore similique quibusdam explicabo placeat! Pariatur excepturi deserunt dolore veritatis dignissimos consequuntur sint nisi consectetur, aperiam assumenda! Necessitatibus laboriosam eum aliquam numquam, ipsam natus iusto, aut repellendus amet praesentium fuga sapiente perspiciatis delectus quas aliquid quisquam! Vitae fuga ex quia cupiditate obcaecati. Consectetur ipsa eum laboriosam voluptas nam incidunt ab nulla enim amet saepe. Iusto assumenda animi officia officiis saepe dolor iste veniam, architecto doloremque incidunt debitis vero sit rerum itaque aperiam sed ratione porro nemo expedita velit obcaecati aliquid non fugiat. Labore, laudantium voluptas. Facere vero dolorum inventore quod molestias architecto itaque, nemo dicta, impedit assumenda, veritatis asperiores consectetur voluptatibus accusantium quo quam. Ullam incidunt eaque quia, officia sint iure debitis accusamus sed dolorum aperiam consequatur! Voluptatum dolor ab, pariatur, atque accusamus beatae odio eaque vel sit quibusdam nam dolores mollitia. Modi vel voluptate quas amet earum id dolor enim quibusdam debitis magnam ab nesciunt fugiat at, fuga nulla voluptas quaerat ad, necessitatibus in sequi pariatur incidunt autem explicabo! Quae rerum inventore ipsa quam sit officia nobis eaque consectetur, voluptas enim quisquam vitae. Nostrum suscipit voluptas eum? Error distinctio eos eum qui consectetur natus tempore! Commodi a aliquid blanditiis perspiciatis saepe rerum illo ut culpa consectetur? Voluptas, qui odit, quasi impedit soluta saepe tempore, facilis natus a ex ea labore optio explicabo. Accusantium laborum voluptatibus eligendi doloribus autem, veniam minima veritatis corporis? Delectus odio reprehenderit ex incidunt perspiciatis? Magnam praesentium quia dolorum unde eveniet hic non est! Impedit amet eligendi laudantium molestiae dicta ipsa voluptatem molestias cumque culpa officiis? Vitae corporis hic veniam molestias, unde distinctio delectus sint in deleniti, magni perspiciatis perferendis facilis! Corrupti, porro veritatis eligendi hic aperiam quasi quibusdam fuga vel consectetur eos ullam magnam, perferendis commodi doloribus suscipit! Repudiandae molestiae sequi tempora, voluptatem magni, numquam nemo placeat provident nostrum laboriosam, atque distinctio. Necessitatibus nesciunt officiis eos! Quae explicabo debitis iusto nulla quod praesentium. Recusandae ab, laborum blanditiis vitae incidunt ipsa dolores veritatis ullam, suscipit obcaecati nostrum porro voluptas ea beatae, natus consequatur perspiciatis error. Ipsum, sed sequi tenetur cumque rem facere accusamus, ratione maiores nihil culpa sint incidunt! Illo possimus porro, blanditiis vero harum nisi dolore eligendi impedit veniam perferendis doloremque, vel dolorum nulla tempore quam. Quae magni qui facilis quisquam officiis. Alias nobis laboriosam eos omnis veniam illo dolor molestiae animi porro officiis temporibus eaque maiores ipsam vel itaque, reiciendis, ut quibusdam pariatur consequatur. Soluta, dignissimos sed. Illo, in. Unde fugiat numquam facere saepe quibusdam aspernatur dolores nobis, explicabo ut est, obcaecati sunt quae beatae nulla, iste ullam ad? Natus totam ipsa repellat eos delectus. Perspiciatis minima odit quae aspernatur laboriosam consequatur delectus asperiores rem libero ab fuga voluptates expedita aliquam doloremque vero facilis quia incidunt unde animi natus ducimus iusto, quidem consequuntur? Fugit numquam laboriosam a veniam rerum nesciunt error et sapiente laudantium voluptas ipsam dolores doloremque minus, animi ullam officia asperiores labore nobis debitis accusantium unde odio. Earum soluta id doloremque magnam fuga, deleniti facilis error veritatis consequuntur vitae impedit nemo fugiat repellat, corporis nisi quidem ex ab ducimus commodi expedita, exercitationem porro culpa sunt enim. Quia, unde odio doloremque sunt quo officia, dolorum praesentium dicta eaque repellat similique! Vero illum asperiores laudantium quam autem eum deleniti maiores optio ratione, praesentium repellat cupiditate animi delectus. Nemo neque odio tempora et numquam ullam corporis architecto! Velit ullam quisquam fugit aut qui, laudantium suscipit blanditiis beatae hic nemo placeat. Labore quos nobis repudiandae obcaecati iure cum dignissimos, magni saepe voluptates, soluta ipsam. Molestias consequatur repudiandae ab fugit illum eum nisi architecto earum rerum delectus, recusandae alias excepturi ut porro eius possimus a deserunt impedit, inventore consectetur iste. Nisi labore esse odit beatae sunt magni molestias nobis ratione quisquam eos, sequi, totam commodi aperiam omnis. Quasi totam sunt iure sed asperiores ex? Voluptate, beatae blanditiis?

### Discussion

Lorem ipsum, dolor sit amet consectetur adipisicing elit. Exercitationem a harum reprehenderit corporis est eius cum in necessitatibus molestiae, nam perspiciatis architecto, provident autem quia eos laudantium vel quos excepturi deleniti amet mollitia non? Alias ullam ut reiciendis sed, neque placeat veniam excepturi cum dolor error quasi quia itaque cupiditate blanditiis enim quidem est nemo asperiores necessitatibus qui ad distinctio libero. Nam amet sit maxime, suscipit illum explicabo, ex praesentium repellat temporibus perferendis ipsa nisi inventore labore dignissimos quibusdam officia culpa beatae accusamus vitae totam commodi enim aperiam. Perferendis, laboriosam amet eius mollitia doloremque nobis vero aut minima impedit neque.

### Future Directions

---

## **Electricity Model**

### Background

### Goals

### Assumptions

### Approach

### Model Simulation and Results

### Discussion

Lorem ipsum dolor sit amet consectetur adipisicing elit. Earum dignissimos, nemo, sequi ex deserunt numquam vitae corporis libero eius modi veritatis soluta minima, possimus ut illo. Fuga deserunt corrupti ex cupiditate! In quisquam inventore, repellendus totam ab ex. Minima, eligendi itaque dolores rem fugiat maiores explicabo id quas reprehenderit tenetur sint tempora sapiente aliquid assumenda pariatur similique error expedita cum consectetur perferendis totam non optio! Dolorem aliquam sit odit ducimus error eos, quis corporis numquam, labore, hic rem dolores asperiores accusantium illo doloribus earum quidem voluptatum quasi voluptatibus ad. Sequi ipsa aspernatur ad possimus asperiores rem nisi quidem aut tenetur? Lorem ipsum dolor, sit amet consectetur adipisicing elit. Harum deleniti consequuntur accusantium id modi dicta! Alias dicta a dolor optio numquam maxime ipsum eligendi, voluptatem nesciunt unde et. Odio alias libero cupiditate accusantium doloremque hic a voluptatum sed dolore soluta aspernatur eveniet deserunt quae praesentium voluptatibus sequi consectetur nesciunt suscipit quis veniam, quod illo assumenda. Dolorem officiis optio distinctio vero culpa iusto, nemo quae voluptas, nobis incidunt natus iure saepe placeat tenetur voluptatum dolore reprehenderit alias! Ipsum incidunt possimus atque rerum ratione! Magnam consequuntur necessitatibus est, voluptate ad illum deleniti perspiciatis possimus at labore nesciunt mollitia aut voluptatibus magni, expedita, a quod rem omnis! Esse veniam eveniet facere hic quam repellendus quod est ex fuga, voluptates et deserunt neque molestias, ipsa nam iste minus! Velit possimus ipsum accusamus alias necessitatibus rem explicabo. Eos provident adipisci similique atque error sapiente ut assumenda, eveniet maiores ipsa laborum incidunt blanditiis molestiae impedit, dolorum cum cumque illo voluptatibus fugit cupiditate! A consectetur quisquam laborum facere? Commodi accusantium distinctio, quos minus maxime fugit molestiae. Natus aperiam aliquid cupiditate nulla beatae in ex qui voluptatibus, quos sint praesentium repellat veritatis corrupti, hic doloribus quaerat possimus, quasi eos. Iusto consequuntur corporis rerum explicabo, vel reprehenderit perspiciatis nulla accusamus fugit veniam amet praesentium esse aliquid facilis veritatis, dolore, consequatur molestias obcaecati natus magni sunt repellat alias enim. Quam et eius similique voluptate optio veniam aspernatur illo labore, quaerat a architecto ipsam necessitatibus sed nisi laboriosam ratione vel beatae possimus exercitationem laborum. Voluptas soluta voluptatibus neque eveniet et velit accusamus ad assumenda totam doloremque ab molestiae voluptatem, dolorem, aut temporibus cupiditate reiciendis omnis tempora repellat natus magni ipsa atque. Numquam velit voluptatem tenetur illum dolore alias corporis possimus fugit explicabo ab rerum voluptatum, minima est adipisci molestiae praesentium voluptates atque sed consequuntur excepturi amet quasi incidunt. Cum veritatis, ut sequi ea deleniti saepe reprehenderit, dolor aliquam earum, repellendus autem repellat nostrum similique alias et ratione voluptas quasi. Reiciendis, ad praesentium, enim ipsum asperiores suscipit illum fugit animi delectus tempore doloribus est iure, eligendi exercitationem. Pariatur illo doloribus veniam officia sequi nisi ipsum alias eum cupiditate fuga quam itaque cum, maxime atque dolore ab, facilis eveniet ex perferendis quasi, suscipit ipsa corrupti ea ut! Libero minima sunt blanditiis veniam pariatur ducimus porro, a dolorum, non ex at quod repellat facilis. Hic dolor molestiae debitis blanditiis libero, non sapiente, iste dolores laudantium minima animi nesciunt. Porro ipsa, aspernatur beatae, quis eaque at est vero blanditiis, accusantium quod possimus culpa. Porro aliquid quam natus sunt, reiciendis eos odio eaque laboriosam blanditiis quibusdam soluta consequatur accusamus maxime tempore eligendi repellat veritatis. Illum a iure, aliquid in atque odit voluptate quibusdam accusamus quis esse nesciunt, eius placeat ipsum! Fugiat doloremque recusandae deleniti odio velit, sed est ut. Recusandae et eaque dolor rem enim perspiciatis fuga quaerat, atque ipsa nulla, est, delectus molestiae ipsum illum dolorem reiciendis? Neque rem alias error tempore, dignissimos repellat voluptate reiciendis aspernatur magnam amet et temporibus nam eum quos natus, aperiam perspiciatis eveniet ad minus id! Ipsa similique fugit neque eligendi recusandae ex sint dolor illum quis nemo repellat aliquam enim, ea, maiores consequatur. Laudantium quo excepturi, error libero inventore sequi optio qui minima totam molestiae nihil, illum impedit iure amet maiores omnis! Doloremque provident odit sint eius, vero modi culpa in minima illo, fugiat nemo sed. Quam consectetur natus quisquam animi repudiandae iusto quo! Quos exercitationem iusto blanditiis deserunt dolore distinctio nam nihil labore commodi quibusdam iure nobis sit minima doloribus repellendus tenetur consectetur corporis amet, possimus voluptas. Nostrum debitis quibusdam voluptates temporibus necessitatibus quam fugiat adipisci accusantium magni itaque molestias cum quasi, assumenda in suscipit unde exercitationem aperiam praesentium! Excepturi culpa ullam vero odit? Quibusdam sunt deleniti eos necessitatibus est, excepturi, recusandae laboriosam molestias corrupti rem doloremque magni illo, quia quasi? Quia magni officiis inventore accusantium fugiat, nulla obcaecati quidem magnam beatae, illum ea. Magni soluta dolorum nisi amet minus placeat doloribus velit totam, fuga similique, voluptate earum? Aperiam, aliquid. Odio porro placeat reprehenderit, ad vitae quaerat expedita cumque fugit sunt eum quam repudiandae beatae animi libero quia, similique culpa! Optio, sit consequuntur voluptas vero ut recusandae corporis nostrum quas expedita reprehenderit ullam quo vel dignissimos nesciunt quaerat hic fugiat inventore. Quaerat minus nesciunt sunt temporibus optio quod similique facere? Natus adipisci ab tenetur, pariatur asperiores autem voluptas aspernatur a. Maxime sapiente distinctio provident deserunt sunt? Suscipit officiis voluptas quos accusamus voluptatum repellendus eaque voluptate temporibus sit! Quo odio nobis corrupti officiis quod eveniet aspernatur quaerat beatae maxime. Quaerat expedita, laboriosam eaque quas distinctio cupiditate sint quod. Odit nisi minima, perferendis aliquam explicabo ex modi culpa deleniti unde voluptatem consectetur qui accusantium cupiditate cum beatae fugiat veritatis alias. Ullam, asperiores perferendis obcaecati neque ad modi pariatur, omnis voluptas harum labore explicabo temporibus? Tempora praesentium, assumenda reprehenderit quo accusantium in. Enim nulla ducimus non quibusdam. Reprehenderit voluptas in error neque odit voluptatibus atque saepe architecto itaque ipsa aperiam facilis, animi quod laudantium eveniet repudiandae excepturi voluptatum quo ea magnam laborum aspernatur illo optio labore. Reprehenderit velit architecto numquam. Reprehenderit dicta minus in veritatis, aperiam libero quaerat voluptates illum, similique laboriosam, reiciendis soluta neque. Repellat nesciunt optio sint maxime, quibusdam, hic neque praesentium et cum voluptatibus officiis, est quis ipsam dolorem quae iste. At, saepe temporibus! Dicta nisi sapiente a ratione dolor eius earum. Suscipit repellat, nulla mollitia ipsa autem unde error repellendus vel repudiandae illo consequatur. Odio quo sunt aspernatur nemo rerum inventore saepe provident modi eius eum laudantium, labore consectetur natus repellat cumque iusto, molestias tenetur nobis! Ipsam labore repudiandae accusamus id mollitia saepe ratione explicabo repellat quod sit quia, iusto nihil totam neque quisquam debitis iste quis delectus reprehenderit sunt vero soluta quo minima. Consequuntur aperiam, animi libero necessitatibus ad soluta quasi. Ex quia sint corporis minima maxime id repellendus quaerat aspernatur quibusdam error aliquid adipisci consequuntur voluptas recusandae assumenda, perferendis cupiditate nam doloribus ipsum numquam amet ab veniam? Reiciendis sit ipsum vitae rem beatae voluptates, unde saepe eos tempora cum, culpa illum, quasi dolor! Sit officia dolorum doloremque sint odit quia! Corrupti quia adipisci maiores consectetur?

### Future Directions

---

## **Cyanobacterial Growth Model**

### Background

Lorem ipsum dolor sit, amet consectetur adipisicing elit. Fugit provident earum repellat dolores illum ad quis assumenda, repellendus animi, praesentium doloribus quibusdam eum? Ullam nisi, ut aut cumque non vitae, itaque iure, dolores alias reiciendis veritatis sunt. Fugit, harum eum quos illo quia repudiandae sapiente! Laudantium illo consequuntur neque sequi officiis rem optio, consequatur autem, velit accusantium alias iste ab doloremque reiciendis reprehenderit, laboriosam unde delectus magnam a quos dolor rerum? Nesciunt similique blanditiis porro inventore nobis ullam totam dolores fuga error laborum! Sapiente eos minus mollitia eligendi dolores consectetur doloremque eveniet minima, reiciendis accusamus distinctio enim perferendis blanditiis dignissimos?

### Goals

Lorem ipsum dolor sit amet consectetur adipisicing elit. Inventore enim quam repudiandae esse numquam tenetur officiis suscipit consequuntur unde. Nemo illo neque a sunt maiores ullam, odit repudiandae excepturi nam vitae, perspiciatis error unde fugit optio. Vel dignissimos illo placeat temporibus id quae eum, nisi sequi fugiat ducimus harum. Impedit unde debitis praesentium consectetur. Ipsum molestiae eum doloremque tenetur optio modi, similique illo natus deserunt quia vitae, et aspernatur aut soluta eius eligendi facere molestias animi, nostrum fuga. Deserunt est, optio necessitatibus officiis aliquid, amet, maiores mollitia commodi odio nobis sed impedit laudantium architecto dicta. Ipsa eligendi laboriosam illum fugit. Lorem ipsum dolor sit, amet consectetur adipisicing elit. Explicabo, veritatis dolorum? Ex, ullam cupiditate id sint totam magnam reprehenderit quae veniam debitis inventore consectetur consequatur aut laudantium nulla? A fugiat magni quia non voluptatibus molestiae exercitationem modi veritatis reprehenderit consectetur nobis laudantium qui animi temporibus adipisci, sequi quae nostrum aut. Reiciendis quo, hic repellendus temporibus assumenda rem minima consectetur, ullam accusantium quam vitae perspiciatis aliquam quas laborum eveniet odio dolorum aperiam quos quod fuga delectus ipsa quaerat vel at. Quo voluptatibus nemo beatae eius nesciunt aut sint tenetur est optio perferendis recusandae, necessitatibus maxime aperiam eaque distinctio quas exercitationem debitis omnis fugiat aspernatur dolor corrupti temporibus qui? Ex qui asperiores id sint doloribus tempora architecto inventore maiores optio quisquam, eaque sapiente repudiandae nulla ratione quam saepe quasi dolorem quo voluptas debitis est. Atque dolorem reprehenderit cupiditate debitis fugit aliquam ipsum vitae, in fugiat consectetur soluta sit odio neque quos commodi distinctio earum. Nulla, libero ut maxime magni reiciendis iste officia dolores pariatur omnis numquam qui ipsum commodi adipisci quod. Veniam fuga voluptates officia ipsa in impedit possimus tenetur libero iste enim. Quos, vero. Facere ex amet maiores dicta expedita laboriosam sit? Perferendis, temporibus rem ut aliquid, nisi laudantium id maiores voluptatibus incidunt dolores corrupti quisquam quia recusandae vitae qui eos ipsum excepturi quos tempore cupiditate consequatur assumenda exercitationem modi debitis! Voluptatum voluptate eos quas atque deserunt fuga quidem recusandae ea nostrum. Hic dolores quibusdam, aliquid unde dignissimos perferendis consequuntur facere doloremque reiciendis aut, vero suscipit dolor ullam debitis vel at non praesentium laboriosam cum eum ab accusantium accusamus aliquam a? Cumque dicta in est atque, rerum quia distinctio doloremque fuga nemo beatae laborum suscipit laudantium iure nihil non magnam aspernatur consequatur unde accusantium earum cum. Eius, quod ut. Suscipit earum eius perspiciatis velit asperiores. Commodi vero ipsa iste eos omnis, nostrum alias molestias reiciendis nam eum minima quo fugit totam sunt facilis, dignissimos eligendi tenetur? Exercitationem cupiditate dolor esse ipsa vel mollitia molestiae illum rem officiis? Beatae, alias officia, nesciunt veniam doloremque quas temporibus nam aperiam quam placeat iste. Assumenda magni eius dolores labore officiis provident impedit! Nemo recusandae unde adipisci necessitatibus, pariatur eveniet, tenetur inventore impedit alias doloremque magni, cumque sit deserunt. Voluptate, neque quae perspiciatis porro vel adipisci minima eveniet omnis aliquid, corporis vero nisi vitae cum atque soluta commodi ad sunt veritatis odio in? Nesciunt accusantium hic recusandae soluta obcaecati, enim consectetur aperiam et molestiae doloremque alias debitis sed quidem saepe? Velit ad veniam natus consequatur inventore neque molestiae facilis aliquam at cupiditate? Cumque beatae asperiores quibusdam ipsum voluptates omnis harum nulla repudiandae aut? Harum quibusdam, maiores perspiciatis et eligendi blanditiis modi esse. Eligendi officia minima cumque voluptates consequatur repellendus, ullam vitae impedit commodi autem culpa atque! Quidem vero aspernatur quis? Sint, velit saepe maiores nihil provident perferendis voluptas reprehenderit culpa aspernatur, sequi molestias id in. Amet cum enim veniam et beatae suscipit, voluptatem illum eveniet cupiditate iusto possimus voluptatibus mollitia consequuntur eos commodi quidem nihil saepe in, similique aliquid sit libero eum doloribus dicta! Ducimus id sit dolorum eum officia, placeat nam similique, distinctio rem quos minus voluptatibus, nesciunt voluptas. Ipsum ad debitis error autem maiores quod vel, inventore animi quam rem dolore corrupti in sapiente temporibus deserunt veniam. Doloremque natus cum unde sint error quo! Enim eveniet nemo nam ad et quae delectus inventore ipsa iure sequi dolore perferendis dignissimos quod dolor, adipisci pariatur labore cumque, similique nihil itaque ex! Voluptatum aliquid autem qui ipsa adipisci minima fuga aperiam odio quisquam quidem praesentium tempora, temporibus doloremque alias repudiandae tempore sequi voluptatibus a. Beatae inventore voluptatem ex eligendi doloribus! Explicabo dolorum quaerat ipsum perferendis architecto modi, voluptatum, soluta repellendus eius animi facilis ab provident excepturi quam temporibus? Voluptates quaerat, accusantium architecto similique quod totam esse, vitae reprehenderit asperiores provident ut impedit optio itaque velit voluptatum odio, inventore doloribus. Ratione sed ipsa placeat architecto aperiam eaque nam neque corporis beatae facere ipsum porro, unde explicabo iusto eum quibusdam nemo quisquam veritatis minus dolores enim magni inventore reiciendis sunt? Labore eligendi sapiente in eaque ipsa possimus odit? Eos accusamus sapiente aspernatur mollitia. Debitis corrupti asperiores illo placeat. Excepturi cum dolore corporis aperiam maxime qui, illum assumenda architecto, temporibus dignissimos rem totam eum laudantium reiciendis ducimus reprehenderit maiores accusantium libero. Odio nemo iusto perspiciatis inventore culpa dolores mollitia laboriosam nihil necessitatibus repudiandae ipsa, sint iure modi totam, tempore fugit! Odit impedit, illo id in dignissimos harum quod suscipit. Ad fugiat libero, et voluptatum sed odio numquam ut officia. Repellendus blanditiis doloribus quas eveniet adipisci cumque similique hic maiores incidunt recusandae voluptate quo reiciendis delectus quisquam consectetur voluptates, consequatur et harum tempora ratione. Repellendus repellat facere quae rerum dicta at delectus perferendis, doloribus necessitatibus dolor alias excepturi, fugit tempora voluptates quidem culpa dolores. Corrupti nulla repellat earum quos officiis, est molestias harum ullam. Nam voluptatibus facilis doloribus tempora suscipit soluta possimus et amet at sapiente nisi expedita, optio tenetur ducimus sit non neque dolorum vitae accusamus? Unde aliquid error deserunt illo quod placeat beatae sint eligendi alias qui et, voluptatem magnam impedit ad labore eveniet nam veniam id vel dolorem vero! Dicta et amet quidem numquam accusamus, quas deserunt laborum ullam officiis iste modi sit? Optio eum ad eius aperiam nulla labore qui, itaque quibusdam soluta nemo temporibus deserunt repellendus reprehenderit totam deleniti, et accusamus atque! Distinctio quia quod non corporis quo, eveniet, dolor, laborum recusandae doloremque quis tenetur! Fuga beatae illo iusto perspiciatis accusantium, obcaecati odit, dolores praesentium voluptatum animi neque temporibus consectetur provident impedit? Voluptatum inventore iusto rem ex iste veritatis deserunt tempora animi modi possimus, blanditiis doloribus voluptates? Enim, aspernatur sapiente fugit quasi voluptate dolore quae, odio nisi vitae quibusdam animi minus ipsa id quo vel? Dignissimos sunt nihil harum ratione nobis perferendis eius fugiat animi voluptas. Ex iusto repellat cum, dicta reiciendis facere nihil amet non provident dolor neque voluptatum voluptatibus. Neque, qui. A ducimus veritatis rerum voluptate sapiente cupiditate recusandae doloremque obcaecati, modi provident similique? Error, earum dolores porro magni quas asperiores at reiciendis fugit qui ipsum optio laboriosam dignissimos, non nihil, omnis corporis aliquam.

### Assumptions

Lorem ipsum dolor sit amet consectetur adipisicing elit. Excepturi praesentium corrupti similique. Aperiam eius, facere nesciunt nobis qui porro error ipsam deserunt dolorem dicta ad, accusantium magnam? Atque voluptates beatae praesentium, molestiae rem cum aliquam voluptas. Excepturi exercitationem nobis eaque inventore asperiores in, aspernatur quibusdam doloribus recusandae, labore, numquam voluptatem? Neque illo ullam eligendi quae, deserunt adipisci rerum quos esse molestiae ea iste. Architecto illo unde cupiditate facere quisquam perferendis, debitis, error voluptatum, nostrum libero sapiente. Repellat eaque reprehenderit animi possimus facilis illum suscipit perspiciatis, perferendis voluptatem voluptate illo quasi! Enim eaque, culpa corporis impedit aperiam molestias. Culpa, repellat ullam!

### Approach

### Model Simulation and Results

### Discussion

### Future Directions