Experiment
Endogenous Induce&Resistance Selection
Here, we want to test the resistance against tetracycline of Alk-Sensor induced by endogenous alkanes.
We add alkanes outside the cell to test the function of TetA:
Construct:
Left: No.60: TetA downstream of Palkm and AlkR downstream of J23103, are cloned into vector PSB1C3 and there’s a alkane producing vector PSB3K3.
Right: No.61: We construct a blank vector PSB3K3 without NPDC and AAR as blank control, and the other vector PSB1C3 is the same as No.60.
Characterization:
Strains of No.60 are cultured in LB medium for about 12 hours. Add 17μg/mL Kan, 12μg/mL Cm and 12μg/mL Tc respectively. After that, inoculate into 3 ml LB medium for an overnight cultures at 37 ℃ with 220rpm shaking.
Result:
Alkane producing E.coli can grow in tube but blank vector E.coli cannot grow .
Conclusion:
Synthesized alkanes induce the expression of alkR, which enables E.coli to survive under the pressure of Tetracycline.
Thus, we can get a 3×3 matrix, with promoter strength from weak, medium to strong, and plasmid copy number from small, medium to large.