iGEM 2023 — Team unc-chapelhill

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## NOTEBOOK

## Weekly Notebook

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## 6/19 - 6/23

Bacterial transformation training

* Plasmid transformation
* Cell pre-culture
* Plasmid extraction
* PlaLarge culture

## 6/26 - 6/30

* Electrodeposition chitosan hydrogels
* Prepare plasmids for sequencing

## 7/10 - 7/14

* Electrode cleaning training
* Cyclic Voltammetry training

## 7/17 - 7/21

* Chitosan hydrogel: 1% v/v acetic acid + 1% w/v chitosan
* Chitosan hydrogels with purple dye electrodeposited onto gold electrodes with chronoamperometry
* Soaked in pH 7 and pH 3 for 30 minutes

* Dye concentration was not high enough to measure

## 7/24 - 7/28

* Assembling enzyme fuel cells training

* Enzyme concentration: 1.13 mg/mL

* Power Test with GDH & ferricyanide

## 7/31 - 8/4

* Repeat power tests with higher concentrations of enzyme (7 mg/mL)

## 8/7 - 8/11

* Repeat power tests with higher concentrations of Ketjen black

* Connected enzyme fuel cells in series to increase power

* Tested BLE device
* Gel Electrophoresis of PQQGDH + pTRC vector

## 8/21 - 8/25

* PQQGDH expression
* Pyocyanin power testing

## 8/28 - 9/1

* PQQGDH transformation with PTRC vector into BL21 DE3 E. coli

## 9/4 - 9/8

* Pre-culture
* Overnight large culture
* Large culture harvest
* DC protein assay

## 9/11 - 9/15

* Power Test: PQQGDH & Ferricyanide
* PQQGDH protein purification with Histag affinity column
* Pyocyanin arrives!!

## 9/18 - 9/22

* PQQGDH protein purification with cation affinity column
* Power Test: PQQGDH & Pyocyanin

## 9/25 - 9/29

* Power test results analysis

## 10/2 - 10/6

* Activity testing for pyocyanin and PQQ

## 10/9 - 10/12

* Wiki finalization

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## ENGINEERING

#### Silver Medal Criterion #1

Hello! World! at least one iteration of the engineering design cycle. This achievement should be distinct from your Contribution for Bronze.

If you plan to show engineering success by creating a new Part that has been shown to work as expected, you must document your contribution on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page) for your team to be eligible for this criteria.

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Please see the [2023 Medals Page](https://competition.igem.org/judging/medals) for more information.

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## EXPERIMENTS

# Wet Lab

## 1. PQQGDH Production:

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pTRC99a vector containing the pyrroloquinoline quinone glucose dehydrogenase (PQQGDH) gene was transformed into Escherichia coli BL21 DE3 cells. A mixture of colonies was incubated into 3mL LB Media (Amp 100µg/mL) for 24hrs at 37℃. One percent of preculture was inoculated into 4, 100mL Flasks of LB Media. Large cultures were cultivated at 37 ℃ until the OD600~0.5 at which 200mL IPTG was added. Protein expression continued at 30 ℃ overnight. Cultures were centrifuged and the wet cells were resuspended in buffer (10mM potassium phosphate buffer;PPB pH 7.0) and lysed twice via French Pressure (Glen Mills) at 2000 psi for 30 seconds. Following centrifugation, ultracentrifugation, and dialysis overnight (10mM PPB pH 7.0) cell lysate was purified via cation exchange chromatography (Resource S 1mL, Elution Buffer 10mM PPB. 500mM NaCl, pH 7.0).

## 2. Electrochemical Analysis:

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### Electrochemical cleaning:

Bare gold disc electrodes were polished (0.05 µm alumina powder), chemically washed (50 mM KOH
Solution (1:5 300mM KOH:H2O2) soak for 45 Min), and electrochemically washed (Cyclic Voltammetry
(CV); -1.2 to -0.2 V (50 V/s; 15 cycles) in 50 mM KOH.

### Enzyme immobilization:

3 μL pf glucose dehydrogenase (GDH) mixed with 22 μL Ketjen black (KB) was prepared for the
anode while multicopper oxidase (mCOP) mixed with KB at the same volumes was prepared for the
cathode. Clean gold disc electrodes were coated three times with 7 μL of the corresponding
enzyme/KB mixture with 45-minute drying periods between each coat. This was followed by 1-hour
glutaraldehyde deposition and 20-minute TRIS soak.

### Ferricyanide Power Test - Version 1:

Pairs of PQQGDH immobilized anode and MCO immobilized cathode were placed in electrochemical cells with 20mM glucose and 100 mM PPB. The electrochemical cell was connected to a resistor box at 1MΩ resistance. Open-circuit potentiometry (OCP) measured the drop in potential between the anode and cathode (mV) over time. This experiment was repeated for concentrations of ferricyanide at 0 μM, 25μM, 50μM, 75μM, and 100 μM.

![](https://static.igem.wiki/teams/4893/wiki/experiments/anodecathode.png)

PQQGDH anode and mCOP cathode demo

### Pyocyanin Power Test - Version 2:

Pairs of PQQGDH immobilized anode and MCO immobilized cathode were placed in electrochemical cells with 100 mM PPB. Open-circuit potential (OCP) was measured by the decrease in the potential between the anode and cathode (mV) over time. Pyocyanin was added to the buffer solution at 0 μM, 25μM, 50μM, 75μM, and 100 μM and tested without glucose. Then, each concentration of pyocyanin was tested with 20 mM glucose in the solution. The electrochemical cell was connected to a resistor box set at 1 kΩ and then raised to 100 kΩ and 1 MΩ.This produced OCP data for concentration of pyocyanin 0-100 μM both with and without glucose and at three different resistances.

## 3. Activity Testing

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Clean bare gold disc electrodes were placed into a solution of 10 mM PPB with 1mM Ca2+ ion and 100 mM Cl- ion supplement. Cyclic voltammetry was run -0.5 to 0.3 at 50 mV/s for 2 cycles. Argon gas was released into the solution to pump oxygen out of the system. Pyocyanin at 100 μM concentration, PQQ at 1 μM concentration, and the two reagents combined were tested.

## 4. Hydrogel Electrodeposition

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Hydrogel solution was prepared with 1% w/v chitosan and 1% v/v acetic acid. Clean gold disc electrodes were placed into the hydrogel solution. Chronoamperometry was run for 7 minutes to electrodeposit the hydrogel onto the electrodes.

![](https://static.igem.wiki/teams/4893/wiki/experiments/hydrogeldemo.png)

# Dry Lab

Data sheets from the pyocyanin and PQQGDH power test were k-clustered by voltage, where k=6, in order to allow for the data to naturally separate into groups corresponding to their resistance level. K was chosen greater than 3 although there were 3 resistances to account for noise and outliers in the data, even among clusters. Each graph was then visually expected to decide which cluster medians should be counted into the mean of interest. For example, if the 10 ohms time section had 3 clusters, the mean of the 3 medians of the 3 clusters would be considered as the central tendency of the 10 ohms section. In other words, we visually checked the graphs, grouped similar clusters together based on when resistance was changed, and then found the average of their medians to represent the central tendency for each section.
When k=6 K-means could not adequately cluster the data, the data was manually clustered into other k values until an adequate value was found—one that could best represent a series of times while including as few outliers as possible. Medians, variances, and sample sizes of each cluster of interest were recorded. Next, these data were used to create graphs and run statistical tests, principally least-squares linear regression, ANOVA, and post hoc Turkey HSD tests.

Recommendations for future data collection:- Measure each resistance for the same amount of time
- Mark at what time you changed the resistance
- Change the voltage unit to a smaller one

Mean Voltage of Every Trial in the format:
GLC or NGLC signifies glucose or no glucose,
25, 50, 75, or 100 signifies the pyocyanin concentration,
R1, R2, or R3 signifies the resistance level (low, medium, and high respectively),
E1, E2, E3, and C, signify electrode number or control

Linear Regression was done to determine if a trend exists between the voltage and any of the variables. Control data was not considered in regression so as to not alter the results. There was a significant relationship between resistance and mean voltage (p < 2.139e-05, R2=0.269) but not a significant relationship between the concentration of pyocyanin and mean voltage (p>.68). This suggests that pyocyanin concentration did not appreciably alter the voltage differences.

ANOVA was done to compare means between concentrations of pyocyanin, resistance levels, and electrodes, and whether glucose was present or not. Control data was not included so as to not influence the data except when doing ANOVA on electrode number. There was no difference in means between glucose and no glucose (p> .718). There was also no difference in mean voltage between pyocyanin concentrations. Along with the linear regression results, this further suggests pyocyanin concentration didn't significantly influence mean voltage differences. There was no difference in means between control and electrodes 1 and 2. However, Electrode 3 was significantly different than both the control and the other 2 electrodes (p < 0.0000778). This informs us the experimental conditions had no effect on the mean voltage when measured with those two electrodes, but there was a difference when measured with electrode 3. Electrode 3 was significantly different than electrodes 1 and 2, suggesting there is a bias in an electrode, as the electrodes were not designed to measure the voltage differently.

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## RESULTS

### Our Experimental Question:

#### If pyocyanin is present, PQQ-Glucose dehydrogenase (PQQGDH) can utilize pyocyanin as an electron acceptor which will produce an electrochemical current response in a concentration dependent manner. If the pyocyanin is absent from the system, there will not be adequate electron transfer and no current will be produced in the system.

## Enzyme Fuel Cells

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In an enzyme fuel cell there are two electrodes - an anode and a cathode - that are placed in a solution. The anode and cathode create a complementary electrode pair that each have immobilized enzymes on their surface. At the anode, the immobilized enzyme oxidizes the substrate in the solution. With the assistance of redox mediators, the electron is transferred to the surface of the anode, creating flow of current. Meanwhile, the enzyme at the cathode causes the reduction of surrounding oxygen molecules and transfers electrons back into the system to maintain the Law of Conservation of Mass. When current from the enzyme fuel cell is connected to a resistor, which generates a voltage. With increasing current and constant resistance, an increased voltage differential between the anode and cathode becomes more prominent, as represented by Ohm’s Law:

*V (voltage) = I(current) x R(resistance)*

In this closed electrochemical cell, we employ PQQGDH as the anode, which catalyzes the oxidation of glucose to gluconic acid, and MultiCopper Oxidase (MCO) as the cathode to reduce oxygen. We use a potentiostat to measure the voltage drop to quantify the current being generated by the reaction, indicating the electrons are being transferred to the surface of the electrode by pyocyanin. To evaluate the efficacy of this closed electrochemical cell, we utilize a potentiostat to quantify the voltage drop, and therefore the resulting current dampening, generated by this reaction which confirms our hypothesis that pyocyanin may be a sufficient electron mediator of this system.

![](https://static.igem.wiki/teams/4893/wiki/anodecathode.png)

PQQGDH anode and mCOP cathode demo

## Test Results

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#### Ferricyanide and PQQGDH Power Test

Goal: Verify the concentration dependency of PQQGDH by utilizing ferricyanide as a known electron mediator.

![](https://static.igem.wiki/teams/4893/wiki/results/singleenzymetest-update.png)

Figure 1. Concentration dependent power generation of ferricyanide utilizing PQQGDH.

Due to shipping delays in our pyocyanin, the first weeks of wet lab were spent using ferricyanide as an alternative electron acceptor. The results display our proof-of-concept that increasing concentrations of ferricyanide at clinically relevant pyocyanin concentrations, 0-100 μM [1], there is a voltage drop across the anode and cathode, and therefore power is generated. These results guarantee that electron acceptor concentration can be monitored by the change of open circuit potential, in the presence of an enzyme with its substrate.

#### Pyocyanin and PQQGDH Power Test

Goal: Validate that pyocyanin is able to accept an electron from PQQGDH.

See Dry Lab section on our Experiments page for details on data cleaning and analysis.

Three pairs of anodic electrodes (PQQGDH) and cathodic electrode (MCO) enzyme fuel cell pairs were tested with and without glucose at 100 μM pyocyanin concentrations and at three different resistances It is expected that without glucose, the PQQGDH cannot perform an enzymatic reaction and no voltage drop will occur.

![](https://static.igem.wiki/teams/4893/wiki/results/screenshot-2023-10-11-at-9-44-35-pm.png)

Figure 1. Mean Voltage for Electrode Pair 1.

Enzyme fuel cells with PQQGDH on the anode and mCOP on the cathode were placed in a 10 mM PPB buffer and 100μM pyocyanin solution. The power generation without glucose appears to be higher than with the 20 mM glucose present, especially for R3 at 1 MΩ resistance.

![](https://static.igem.wiki/teams/4893/wiki/results/fig3-results.png)

Figure 3. Mean Voltage for Electrode Pair 3.

Enzyme fuel cells with a bare gold disc electrode on the anode and mCOP on the cathode were placed in 10 mM PPB buffer solution and 100μM pyocyanin. The power generation without glucose appears to be higher than with glucose present for all three resistance values. This electrode pair had the highest voltage reached at 70 mV.

![](https://static.igem.wiki/teams/4893/wiki/results/screenshot-2023-10-11-at-9-48-03-pm.png)

Figure 2. Mean Voltage for Electrode Pair 2

Enzyme fuel cells with PQQGDH on the anode and mCOP on the cathode were placed in a 10 mM PPB buffer and 100μM pyocyanin solution. The power generation without glucose appears to be roughly the same at the R1 and R3 values, and significantly higher with glucose at the R2 value.

![](https://static.igem.wiki/teams/4893/wiki/results/control-results.png)

Figure 4. Mean Voltage for Control Group.

Enzyme fuel cells with no enzyme on the anode and mCOP on the cathode were placed in 10 mM PPB buffer solution and 100μM pyocyanin. The glucose-absent cells showed no reaction occurring, while the glucose-present cells showed a voltage drop despite no PQQGDH in the solution.

![](https://static.igem.wiki/teams/4893/wiki/results/table1-results.png)

Table 1. Summary Table of PQQGDH and Pyocyanin Power Test for 1Ω resistance.

Means calculated from Dry Lab analysis as explained in Experiments. Each “pair” refers to an anode and cathode in one enzyme fuel cell. Table 1 highlights the mean values calculated from our highest resistance value at 1MΩ. This resistance value was chosen for each anode/cathode pair because it displayed the clearest distinction between enzyme fuel cells with and without glucose present.

## Discussion

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In our pyocyanin power test experiments, the expected result was to detect a higher voltage drop when the glucose is present due to the electron transfer reactions that occur with PQQGDH and pyocyanin. At present, our results are inconclusive due to the increase in voltage for the fuel cells without glucose compared to those with glucose, as seen for electrode pair 2 and 3 in Table 1. There are a few points of consideration that lead us to think this data is unreliable.

Firstly, the enzymes on the anode were crude enzymes, due to our difficulty in purifying the PQQGDH enzyme. Because this was crude enzyme sample, we cannot be sure how much of this solution matrix is comprised of our target PQQGDH enzyme and that other unwanted proteins may have obscured our results. Secondly, the PQQGDH was not incubated with Ca+ ions to improve the metal-ion coordinate binding between the GDH and PQQ cofactor. Future experiments will incorporate this step. Finally, the control was a clean, bare gold disc electrode. However, a more representative control would be an electrode without enzyme but with Ketjen black. Since ketjen black creates a conductive carbon matrix to immobilize the enzyme onto the surface of the electrode, it limits the electrode surface for electron transfer. It is also interesting that voltage measurements were often higher when the PQQGDH had no glucose to oxidize. This indicates an alternative current source that is generating the power and not the PQQGDH.

These results cannot yet confirm whether or not pyocyanin can act as an electron acceptor for PQQGDH. However, further testing with pyocyanin and PQQGDH is required to understand the thermodyanamic and electrochemical properties of this reaction.

## Cyclic Voltammetry (CV) Activity Testing

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Goal: Determine if the transfer of an electron from PQQGDH to pyocyanin is thermodynamically possible.

![](https://static.igem.wiki/teams/4893/wiki/results/cyclicvoltsresult.png)

Figure 5. PQQ and Pyocyanin CV.

Standard Cyclic Voltammetry is a fundamental electrochemical technique that is able to perform two alternating potential sweeps in a triangular waveform, designated the anodic and cathodic sweeps. The forward oxidative sweep from negative to positive potential creates a positive current, while the reverse reductive sweep creates a negative current. In order for PQQGDH to spontaneously reduce pyocyanin, the oxidation peak of PQQ must have a higher numeric potential than the reduction peak of pyocyanin. Here, we have a CV scan of 100 μM pyocyanin and 50 μM PQQ that displays a higher oxidation peak for PQQ, around -0.15 V, than the pyocyanin reduction peak at -0.25V. These results indicate that it is thermodynamically possible for pyocyanin to be an electron acceptor, without considering factors such as mediator accessibility.

## Chitosan Hydrogels

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Goal:
Demonstrate proof-of-concept to incorporate a chitosan hydrogel matrix to the electrode fabrication.

The chitosan hydrogels with dye were successfully deposited onto the surface of a gold disc electrode. Our pH dependency testing was unsuccessful because the concentration of dye release was too low. Further testing was dissuaded to focus on the PQQGDH-pyocyanin electron transfer mechanism.

![](https://static.igem.wiki/teams/4893/wiki/experiments/hydrogeldemo.png)

## Final Conclusion

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While we were not able to test and validate our sense-and-treat design altogether, we took significant steps in building our P. aeruginosa biosensor. Initial tests of PQQGDH and pyocyanin were uncertain in showing that pyocyanin could be an electron acceptor of PQQGDH. However, the cyclic voltammetry scan proved that this reaction is still possible, and, given more trials with the discussed improvements, results may prove to be more successful. Overall, we completed the beginning stages of building our design and hope to continue this project past the iGEM season to obtain more concrete results.

## Reference

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[1] Alatraktchi FA, Andersen SB, Johansen HK, Molin S, Svendsen WE. Fast Selective Detection of Pyocyanin Using Cyclic Voltammetry. Sensors (Basel). 2016 Mar 19;16(3):408. doi: 10.3390/s16030408. PMID: 27007376; PMCID: PMC4813983.

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