Results of plasmids construction
*n represents for pTrc99k-nahR plasmids.
*G represents for pMW119-Psa1-EGFP plasmids.
We then used the successfully constructed plasmids to examine the effectiveness of the bio-sensor system.
Some possible factors that led to failure
Mistakes in homologous recombination
Unable to recombine the right fragments into the vector.
Mistakes in PCR
During the amplification, the fragments may not be correctly amplified by PCR, so some of the recovered fragments are wrong fragments, which will cause problems when used for vector construction
The pTrc99k-NahR and pMW119-Psa1-EGFP plasmids were co-transformed into Tatumella Citrea and screened by Kanamycin resistance and Ampicillin resistance LB solid plates. The co-transformed strains were inoculated into fresh LB medium, cultured at 37°C until OD600 was approximately equal to 0.6. Different concentrations of salicylic acid (dissolved in absolute ethanol) were added. After 24 hours, observations with fluorescence microscope were used to show the regulatory effect of salicylic acid on the PsaI promoter and it was indicated by fluorescence intensity.
Figure 1. Microscopic view of fluorescence shown by Tatumella Citrea
Table 1.OD600 of Tatumella citrea under different salicylic acid concentration
Table 2.Fluorescence intensity under different salicylic acid concentration
Processed data table 3. Calculated homogenized fluorescence intensity under different salicylic acid concentration
We measured the fluorescence intensity to obtain a quantitative result. The data is plotted as the following: the measured fluorescence intensity is divided by the number of cells (OD600) to obtain a homogenized fluorescence intensity. The fluorescence intensity of all samples added with salicylic acid is subtracted from the fluorescence intensity of the background without salicylic acid. It can be seen that from 1 μM to 100 μM salicylic acid, the fluorescence intensity rises sharply. Under the condition of 1000 μM salicylic acid, the fluorescence intensity decreased, which is presumably due to the influence of salicylic acid solvent (ethanol) on cell growth, or other unknown regulatory effects of salicylic acid and NahR.
Figure 2. Relative fluorescence unit under different Salicylic acid concentration
With the same concentration of salicylic acid, we also measured the change of fluorescence intensity over time. It can be seen from the figure 3. that within 3 to 22 hours after the addition of salicylic acid, the fluorescence intensity increases successively. But it mostly stops increasing at 24 hours.
Table 4. OD600 of Tatumella Citrea in different hours after addition of salicylic acid
Table 5. Fluorescence intenstiy of Tatumella Citrea in different hours after addition of salicylic acid
Processed data table 6. Calculated homogenized fluorescence intensity in different hours after addition of salicylic acid
Figure 3. Relative fluorescence unit with different time after addition of salicylic acid
The mechanism we created did work out well, and the expressions of EGFP protein was detected. This proves that the potential of the bio-sensor system we made is effective, and the GFP protein can be replaced with GADPH and reach the regulatory effect. The two factors we examined are the most convenient, and available test we could conduct. For the concentration of salicylic acid, we found out that at 100 μM, it shows the highst fluorescence intensity. This means that the system works the best in that range of salicylic acid concentration. For the time after addition of salicylic acid, the highst fluorescence unit is shown at 22 hours, the trend is mainly increasing. But after 22 hours, it is decreasing. It is best to leave the mechanism for 15-22 hours after the addition of salicylic acid so it can show the most effective results.
Through this experiment, there are still some work we could do to make our results better. Firstly, we need to find out how temperature, pH or other elements impact upon our results. Secondly, we planned to construct a GAPDH knock-out Tatumella citrea strain to check out if this strain can commensalism with crops.
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