Part:BBa_K3805238:Experience

Applications of BBa_K3805238

To verify the function of this part, we detected the expression of the downstream gene. In our experiment, we used two genes. One was GFP and the other was endotoxin ccdB.

As for GFP, we expanded the culture of bacteria containing plasmid-G, and then divided evenly into several parts. Added mCherry solution to one of them, and made loading tablets and observed the fluorescence under the fluorescence microscope. Then set the concentration gradient of mCherry solution, and measured the fluorescence intensity of GFP produced by bacteria with different mCherry fluorescence intensity by microplate reader. Theoretically, if anti-mCherry pmrB could effectively bind to mCherry, we would observe the green fluorescence and the increasing trend in its intensity. For ccdB, we expanded the culture of bacteria containing plasmid-B, and then divided evenly into several parts. According to the data measured in the previous experiment, mCherry solution with appropriate concentration was added to several of them and incubated for a period of time. The other was added with buffer as control. Applied the culture medium to the culture plates to form single colony. Counted the number of growing colonies and calculated the mortality, so as to obtain the killing efficiency. With the ideal result, the number of colonies was reduced compared with the control group.

We completed the confirmatory expreiments and obtained the expected reults successfully. The green fluorescence was obseved under fluorescence microscope and intensity of GFP was detected on the increase(Figure 1 and Figure 2). Meanwhile, according to the colony numbers, we saw that the colony growth after adding mCherry was worse than adding buffer solution, which demonstrated the killing pathway was effective(Figure 3).

Figure 1 cheaters without mCherry (fluorescence microscope 1000X)

Figure 2 cheaters with mCherry (fluorescence microscope 1000X)

Figure 3 Comparison of growth of cheaters with and without mCherry (Buffer solution added on the left, mCherry added on the right)

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