1. E. Coli response to Theophylline and Caffeine

2. Procedure Using a plate reader absorbance was measured of J11940+J119303, J119140+J119304, J11940 in pSB4c5, J119140 in pSB1a2, K315009, and J100111 in the presence of 1 mM of theophylline and separately in the presence of 5 mM of caffeine. Each combination was tested in the presence of 10 microgram/mL of tetracycline (low concentration), 20 microgram/mL of tetracycline, and no tetracycline. Absorbance was measured every hour for 23 hours

3. Results 15 hour absorbance minus initial absorbance with 1 mM Theophylline NO TETRACYCLINELOW TETRACYCLINEHIGH TETRACYCLINE J119140+J119303 (pT5-TheoRiboSwitch-TetA + high promoter-high c. dog-eCDM8) 0.1790.1620.636 J119140+J119304 (pT5-TheoRiboSwitch-TetA + low promoter-low c. dog-eCDM8) 0.2880.2460.170 J11940 in pSB4c5 (pT5-TheoRiboSwitch-TetA) 0.263n/a0.193 J11940 in pSB1a2 (pT5-TheoRiboSwitch-TetA) 0.278n/a0.214 K315009 (pLacI-RBS-RFP-RBS-TetA) 0.325n/a0.120 J100111 (pT5-RBS-CDM8-pT5-TheoRiboswitch- TetA) 0.5600.523-0.002 All cells except J100111 grew in the presence of Theophylline in all Tetracycline concentrations. It would be expected that the theophylline would have allowed transcription of the Tetracycline resistance in J100111 allowing it to grow in high Tetracycline. Perhaps not enough Tetracycline resistance was made to successfully pump out the high concentration of Tetracycline.

4. Results 15 hour absorbance minus initial absorbance with 5 mM Caffeine NO TETRACYCLINELOW TETRACYCLINEHIGH TETRACYCLINE J119140+J119303 (pT5-TheoRiboSwitch-TetA + high promoter-high c. dog-eCDM8) 0.0120-0.004 J119140+J119304 (pT5-TheoRiboSwitch-TetA + low promoter-low c. dog-eCDM8) 0.0190-0.006 J11940 in pSB4c5 (pT5-TheoRiboSwitch-TetA) 0.173n/a0.008 J11940 in pSB1a2 (pT5-TheoRiboSwitch-TetA) 0.181n/a0.006 K315009 (pLacI-RBS-RFP-RBS-TetA) 0.179n/a0.021 J100111 (pT5-RBS-CDM8-pT5-TheoRiboswitch- TetA) 0.304-0.002 All cells grew in the presence of Caffeine whenever no Tetracycline was present. This shows that the Caffeine is not killing the cells. However, in the presence of Tetracycline only K315009 (the control) grew successfully. These results show that the tetracycline resistance is not being expressed in the other cells.