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
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
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)
Pyocyanin and PQQGDH Power Test
Goal: Validate that pyocyanin is able to accept an electron from PQQGDH.
![](https://static.igem.wiki/teams/4893/wiki/results/screenshot-2023-10-11-at-9-44-35-pm.png)
![](https://static.igem.wiki/teams/4893/wiki/results/fig3-results.png)
![](https://static.igem.wiki/teams/4893/wiki/results/screenshot-2023-10-11-at-9-48-03-pm.png)
![](https://static.igem.wiki/teams/4893/wiki/results/control-results.png)
![](https://static.igem.wiki/teams/4893/wiki/results/table1-results.png)
Discussion
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
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)
Chitosan Hydrogels
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
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
[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.