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Testing for Colour Switch
Once the Opto-FLP and colour-switch plasmids were dually-transfected into BL21 DE3 E. coli cells, two cultures were grown overnight, one in the dark and one in the light (Figure 1). The cultures were then treated with IPTG to induced transcription via T7 polymerase. Samples from both cultures were taken immediately after IPTG treatment (Time 0h), and imaged to test for fluorescence (Figure 2). Both mScarlet and eGFP signal were observed at Time 0h, indicating that there was leaky expression of mScarlet and eGFP. There appeared to be a higher ratio of eGFP to mScarlet in the light treated culture than the dark culture, indicating that there was likely leaky expression of Opto-FLP as well, and it was excising mScarlet from the colour-switch plasmid.
Figure 1: Dual-Transfected Bacteria Under Light Treatment. (A) Dual-transfected BL21 DE3 bacteria were grown on agar plates overnight under light treatment and in the dark (not pictured). (B) When treated with pure blue light, eGFP fluorescence could be observed in the bacterial colonies.
Figure 2: Dual-Transfected Bacteria at Time 0h. The Opto-FLP and colour-switch plasmids were dually-transfected into BL21 DE3 bacteria, and left to grow overnight in a dark environment and light treated environment. The cultures were treated with IPTG and immediately imaged for fluorescence. Both cultures were observed to have eGFP and mScarlet signal, however there appeared to more mScarlet in the dark culture than the light culture.
Samples from the light and dark dual-transfected cultures were taken immediately after IPTG treatment, as well as 1 hour, 3 hours, 6 hours, and 24 hours post IPTG treatment in order to observe any change to the eGFP and mScarlet fluorescence in these cultures. The samples at the various time points were imaged, and the fluorescence was quantified (Figure 3). It was observed that the ratio of eGFP to mScarlet was higher in the light culture than the dark culture, indicating that there was more eGFP and less mScarlet in the light culture, thus Opto-FLP was more effective when the culture was treated with blue light.
Figure 3. Functionality of Opto-FLP to Increase GFP Expression Relative to mScarlett Expression. GFP:mScarlett expression was compared between bacterial cells that contained Opto-FLP and pRSET-FRT-mScarlet-STOP-FRT-eGFP. The functionality of Opto-FLP to increase eGFP expression was tested in the dark (blue) and in the light (orange). Five samples were taken over a period of 24 hours after IPTG induction. Time 0h represents the sample taken immediately after the addition of IPTG.