Team:TU Darmstadt/Notebook/Labjournal/K1497020

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K1497020 - Naringenin Sensor (FdeR) with GFP as Reporter

The Naringenin Sensor (FdeR) is not detectable by sight. For detection, we used several reporters. One of them was GFP, which we got out of the registry (E0040). We cut the E0040-plasmid with XbaI + PstI and cut the Naringenin sensor plasmid with SpeI + PstI. In addition, we dephosphorylized the digestion of the naringenin sensor. Subsequently we ligated the insert into the backbone and behind the Naringenin Sensor using T4 ligase, producing a mixed site between the sensor and the reporter. We transformed the ligation product into Top10 E. coli via heatshock and spread them out on CMP-LB agar plates. We incubated the plates overnight at 37°C and analyzed obtained colonies by colony-PCR and agarose gel electrophores. Positive colonies were inoculated in 5 ml LB with CMP and plasmids were isolated after incubating for 16 h at 37°C. For further verification we send the samples to Eurofins for sequencing.