Team:Lambert GA/Proof Of Concept
PROOF OF CONCEPT
Hypothetically, using the phosphate biosensor cells, aquaponics farmers should be able to identify the phosphate concentrations of unknown water samples with the characterization curve on the Agro-Q App. For instance, if the biosensor cells expressed Y arbitrary units of GFP, the user could find a point (X, Y) in the characterization curve where X would be the corresponding phosphate concentration.
Successful initial characterization trials of the phosphate biosensor, measured by a plate reader, demonstrated response to phosphate concentrations with a similar curve as Lambert iGEM’s deterministic ODE model predicted. This is just the first step in developing the full proof of concept. Establishing an algorithm to compare the arbitrary units of GFP from the plate reader to the measured values from Fluoro-Q will be necessary to correlate Fluoro-Q data to the characterization curve, and then determine the phosphate concentrations from unknown samples. With limited access to a plate reader in the Styczynski Lab at Georgia Institute of Technology due to the COVID-19 pandemic, Lambert iGEM did not have enough time to complete a proof of concept. To continue on the project in the following year, the next step would be to take measurements of serial dilutions of fluorescein with both the plate reader and Fluoro-Q. By comparing known values measured by the two devices, the team will be able to derive a formula to mathematically compare fluorescence data in Fluoro-Q and in a plate reader in order to calibrate Fluoro-Q measurement relative to plate reader measurement. Using the derivation and the relationship between the two devices, a new characterization curve will be constructed based on Fluoro-Q with comparable values between phosphate and fluorescence on both the plate reader and Fluoro-Q. By building this curve and installing the predictive model on the Agro-Q app, interpreting fluorescence data from biosensor cells will become more reliable and accurate. Furthermore, this will allow the team to test GFP expression of biosensor cells under real samples from aquaponics systems.
In order to validate the biosensor’s responsiveness within the phosphate concentration range maintained in Lambert iGEM’s working hydroponics system and fish tank, a series of unknown samples such as waste water from fish will be gathered and measured in biological and technical triplicates by both Fluoro-Q and a plate reader. The phosphate concentrations determined from this fluorescence data will then be compared to the values obtained from commercially available aquarium and water quality tests, which are commonly used by aquaponics farmers due to their cost-effectiveness and quick qualitative results. Lastly, for final verification, the data will be compared to chemical analysis of the samples done by a commercial independent testing lab that provides quantitative data.
| Sample Type | Biosensor Cells (Plate Reader) | Biosensor Cells (Fluoro-Q) | Aquarium Water Test Kit | Commercial Chemical Analysis |
|---|---|---|---|---|
| Tap water (Used to make the hydroponics fertilizer solution) | X | X | X | X |
| Initial fertilizer solution (Before installation in the hydroponic garden) | X | X | X | X |
| Secondary fertilizer solution (After a week in circulation) | X | X | X | X |
| Fish tank water (Before cleaning) | X | X | X | X |
| Fish tank water (After cleaning) | X | X | X | X |
| Water used to fill fish tank | X | X | X | X |
| Phosphate solutions of known concentrations in the range of biosensor detection (Established by earlier testing) | X | X | X | X |
| MOPS media with phosphate biosensor cells | X | X | X | X |
| Plain MOPS media | X | X | X | X |
Table 1. Example spreadsheet for testing the efficacy and accuracy of phosphate biosensor cells, as well as verification of the accuracy of the Fluoro-Q device.