Assembling parts of the CcaS/R and Opto-T7 systems:
Molecular cloning methods were performed to bring together the different gene fragments of the CcaS/R system and the Opto-T7 system.
Fig.1-Molecular cloning
ECFP Characterization:
Since we used ECFP as a reporter protein in our CcaS/R system, we characterized it using the following experiments:
- Protein Precipitation Assay:
For the purification of a protein, one nice method is the Ammonium Sulphate Precipitation method in which different proteins precipitate out from the protein lysate solution at different salt concentrations, i.e. different proteins “salt out” at different salt ((NH4)2SO4) concentrations. To determine the salt concentration at which ECFP protein precipitates, we performed the Ammonium Sulphate Precipitation Assay (Protocol page).
The results for this can be found on the Result page.
Fig.2-Ammonium sulphate protein precipitation assay
- Fluorescence measurement:
To determine the absorption and emission maxima of our fluorophore ECFP, we did a wavelength scan and observed the fluorescence intensity peaks.The fluorescence measurements can be found on the Result page.
Truncation Assay:
To quantify the truncation due to the internal RBS in i2mCherry (Leu), i2mCherry (Ile) and mCherry, we used the following two strategies:
- Firstly, a rough estimate was obtained by comparing the band intensities directly from the gel electrophoresis of the total cell lysate after expressing the protein.
- For a more accurate quantification, we purified the protein using Ni-NTA and measured the fluorescence obtained in all (supernatant, wash, and elution) fractions. In an ideal case, the ratio of fluorescence obtained in the supernatant and elution fractions would give the fraction of protein that was truncated (assuming truncated and non-truncated i2mCherry (Leu), i2mCherry (Ile) and mCherry are equally fluorescent). However, the process of binding of 6xHis to Ni-NTA is not perfect and proteins tend to run off into the supernatant and the washes. To get rid of this experimental error, we performed PAGE with each fraction and divided the total fluorescence of the fraction into two parts - truncated/non-truncated - using the band intensities. The fluorescence for the truncated protein from each fraction was then summed up and divided by the fluorescence of the non-truncated part to get the relative fractions of the two varieties. Concerned results can be found on the Result page.
Fig.3-Ni-NTA protein purification assay
Experiments related to ccdB gene system:
- Growth assay:
Our ccdB gene is under the promoter araBAD. As the name suggests, this promoter is induced by arabinose. To determine the effect of different quantities of the inducer (arabinose) on the amount of gene product, a growth assay was performed. Our gene product being a toxin affects cell growth. Glucose acts as a repressor for araBAD. In order to determine any leaky transcription, we grew the bacteria with the gene system incorporated in the absence of arabinose and in the presence of different concentrations of glucose. Then we grew the bacteria with different concentrations of the inducer arabinose. We then counted the number of CFU and were able to determine the effect of different concentrations of inducer arabinose on the cells and hence could have an estimate of the comparative amount of the toxin produced.The results for this can be found on the Result page.
- Protein extraction and purification:
To procure the pure CcdB L83S protein so as to conduct further studies on it, we carried out a protein purification procedure. We extracted the protein using the Column chromatography method with a CcdA column chromatogram. To confirm whether we were able to extract the protein of interest (CcdB L83S) and have a rough estimate of the purity of the extracted protein, we performed an SDS-PAGE with different elution samples from the column, total cell lysate and the concentrated protein. The results for this can be found on the Result page.
Fig.4-Protein extraction-purification assay
- Protein Thermal Shift assay:
Our protein CcdB L83S is a mutant of the wild type CcdB protein. Thus, it’s stability is expected to be different from that of the wild type protein. The melting point of a protein gives us insight into the stability of that protein. Hence, by determining the melting point of CcdB L83S and comparing it to the wild type protein, we can have a qualitative inference of the stability of our protein. To do this, we determined the melting point of our protein by performing a Protein Thermal Shift assay. The results for this can be found on the Result page.
Fig.5-Protein thermal shift assay
Potential future experiment:
Opto-T7 has never known to be incorporated in B. subtilis. Thus we plan to characterize the system in B. subtilis i.e. we want to determine the optimum conditions for the maximal and most efficient functioning of the gene system. To do this we plan to use spectrometric (OD) analysis. We shall measure the initial OD of the bacterial culture. Then, we shall take this culture into different test tubes (3 mL) and shine different wavelengths of light to each test tube for 1-1.5 hrs. After this, the OD measurement of the culture in different test tubes shall be done. This will help us determine the wavelength of light to be shone on the culture to have the most efficient bacteriostatic effect. We shall also change the exposure time of light and measure the OD after different exposure times to find out the most optimal exposure time.
