Part:BBa_K4836016
Serotonin induced regulatory cassette
This cassette examines the activation of the pLasI promoter when exposed to the Serotonin+LasR complex. Initially, the bacterial SERT membrane protein facilitates the entry of external serotonin into the cell. Subsequently, LasR binds to the internalized serotonin, forming the Serotonin+LasR complex, which activates the pLasI promoter through binding. This activation process will be assessed by measuring the fluorescence intensity of the mCherry reporter gene. An increased fluorescence intensity of mCherry in the presence of the Serotonin+LasR complex, as compared to the uninduced state of pLasI, would indicate the functional capability of the Serotonin+LasR complex to activate the pLasI promoter.
The confirmed plasmid with the desired construct was transformed successfully into E. coli BL21 cells to check the expression and functionality of the regulatory system components. Confocal Microscopy was performed to confirm the fluorescence reporter mCherry present in this module which shows its expression after the Serotonin-LasR complex activates the transcription of downstream genes of the LasI promoter. Expected Results: Either Basal expression or no expression should be observed in the module when no inducer molecule - Serotonin is added. An increase in the intensity of mCherry should be observed when serotonin is added to the culture. We inoculated the primary culture of BL-21 cells which had the confirmed construct and after the OD600 nm reached 0.4 i.e., the exponential growth phase of the cells, inducer molecule - serotonin of 80 $\mu$M concentration (physiological levels of serotonin in IBS-D patients) and 100 $\mu$L volume of this solution is added into 10mL secondary culture. The confocal microscopy was performed at 0min, 40min, and 120mins and observed less significant change in the intensity after 40mins. All the parameters like exposure, emission, and absorption wavelengths were kept constant during the observations. The pictures below depict the intensities observed.
Inference - 1 The above result suggests that the regulatory system induced by Serotonin works well. In the presence of serotonin, as it is clearly visible from the Confocal Microscopy results, the presence of mCherry protein is seen after the addition of serotonin.
Inference - 2 The SERT analog CUW_0748 acts slower than the desired efficiency. As there is no significant increase in the intensities at 40 mins and at 120 mins, we could say that CUW_0748 should be expressed constitutively.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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