Team:IISER-Pune-India/Results/Biosensor
The Biosensor
The Biosensor is a part that has been previously developed by the Beilefeld Team 2015 (BBa_K1758333) as part of their Cell-Free Heavy Metal Detection system. We have characterized this biosensor in E. coli DH5alpha cells using fluorescnece as a readout at different lead concentrations. We have also modelled this biosensor, which can be checked out here
Fluorescence Measurements
The first experiment was to determine whether the part works, and shows significant fluorescence in presence of lead. The negative control is a strain that does not have any GFP containing plasmid (BBa_R0040). The positive control is a strain that has high and constitutive expression for GFP (BBa_J364001)
Excitation and Emission Spectra
This was followed by analyzing the spectrum of the biosensor as it has sfGFP as a reporter gene. The excitation and emission spectrum for the biosensor are shown below:
| Excitation Wavelength Measurement (nm) | 490 - 580 |
| Emission Wavelength (nm) | 513 |
| Emission Wavelength Measurement (nm) | 430 - 520 |
| Excitation Wavelength (nm) | 504 |
Varying the Lead Concentrations
To observe the change in fluorescence intensity, cells with biosensor were grown in LB medium inoculated with varying concentrations of Lead Nitrate (Pb2+). The values that were chosen were - 0,0.001, 0.01, 0.1, 0.5, 1, 5, 10, 15, 20, 30 and 40 (all values are in Micromolar concentration). A detailed protocol is available on the Experiments page.
The above graphs depict individual experiments performed to characterise the biosensor. The error bars indicate the standard error for 3 technical replicate for each sample.
The following graph shows the combined data of the individual experiments. The error bars denote standard error of mean for n = 4 experiments.
The other graph depicts the same data with the x-axis being on the logarithmic scale.
Using modelling, it was determined that the linear range for fluorescence intensity v/s lead concentration is between 0uM and 15uM. The experimental data reflects this, as shown in the graph below.
The survivability and growth of the cells has also been observed by measuring the OD600 values of the cell culture in LB at different Lead concentrations.
The cells containing biosensor were also grown in M9 medi alongwith water samples from different parts of India (see Collaborations for details). The results of that assay are given below. However, no conclusion could be made from the data.
| Sample Name | Location |
|---|---|
| S1 | Mula Mutha River |
| S2 | MIT-ADTBio Pune |
| S3 | REC Chennai |
| S4 | SNU Noida |
| S5 | IISC Bangalore |
| S6 | IISER Tirupati |
| S7 | IISER Bhopal |
| S8 | IISER Kolkata |
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