RESULTS

CHARACTERIZATION

PURPOSE OF CHARACTERIZATION

Based on Lambert iGEM’s ODE model, the 4uM threshold of BBa_K2447000 seems to be less strict than described above. Extracellular phosphate concentrations greater than 4uM, even in the range of 100uM, will still result in minimal GFP expression. To improve upon the existing characterization of the phosphate sensor, Lambert iGEM tested with a greater number of phosphate concentrations from 0 to 100uM in intervals of 20uM.

EXPERIMENTAL DESIGN

In order to improve part BBa_K116404 created by NYMU-Taipei 2008, NUS Singapore iGEM 2017 created part BBa_K2447000 by replacing the weaker RBS BBa_B0032 with a stronger RBS BBa_B0034. This allowed the BBa_K2447000 extracellular phosphate sensor to be much more sensitive to various phosphate concentrations from 0uM to 1000uM.

Through research, the team discovered that typical phosphate levels in aquaponics systems range from 10ppm to 40ppm [3]. Using dimensional analysis, the team concluded that maintaining a phosphate concentration between 50uM and 100uM is ideal for plants and fish to coexist. To add detailed characterization of part BBa_K2447000 on phosphate concentrations specifically targeted for use in aquaponics systems, Lambert iGEM tested GFP expression of the phosphate sensor on extracellular phosphate levels from 0uM to 100uM in intervals of 20uM.

EXPERIMENTAL PROCEDURE

The characterization protocol began with the team’s biosensor cells being grown in chloramphenicol LB for 24 hours and later diluted to an OD₆₀₀ value of 0.4. Then, the cells were pelleted and resuspended into MOPS media, which has minimal phosphate concentration relative to LB. To the 5 mL resuspension, the team added different phosphate concentrations between 0 to 100uM and waited 3 hours for GFP to be expressed. In order to measure the GFP expression, Lambert iGEM used a plate reader from Styczynski Research Group at Georgia Institute of Technology.

CHARACTERIZATION CURVE

Figure 1. Characterization curve showing the relationship between phosphate concentrations between 0 to 100uM and fluorescence/OD₆₀₀ measured by a plate reader.

Figure 2. Prediction of relationship between GFP expression and phosphate concentrations ranging from 0 to 100uM made by deterministic ODE model.

Using data from the plate reader, Lambert iGEM created a characterization curve showing the relationship between phosphate concentrations and fluorescence/OD₆₀₀. For phosphate concentrations ranging from 0 to 80uM, the decreasing trend in fluorescence/OD₆₀₀ closely resembled Lambert iGEM’s ODE model prediction, shown in Figure 2. The fluorescence value for the 100uM phosphate concentration did not match the prediction from the model because the phosphate media was diluted improperly, causing its measured GFP expression to be higher than expected. Due to time constraints in the lab, the team was not able to conduct further testing and decided to use the characterization data for only 0 to 80uM of phosphate.

SAMPLE TESTING

Hypothetically, using the biosensor cells, aquaponics farmers should be able to identify the phosphate concentrations of unknown samples by utilizing the characterization curve. For instance, if the biosensor cells expressed Y arbitrary units of GFP, the user could find a point (X, Y) in the characterization curve in which X would be the corresponding phosphate concentration. This method would be applicable in identifying the phosphate concentration in unknown samples from real aquaponics systems such as a nutrient fertilizer mix, waste water from fish, and even tap water. Lambert iGEM did not have enough time to perform sample testing, but will be conducting experiments next year.

REFERENCES

[1] Storey, N. (2017, December 13). The Most Important Things To Know About Phosphorus. Retrieved October 03, 2020, from https://university.upstartfarmers.com/blog/most-important-things-about-phosphorus.