E. Coli response to Theophylline and

Caffeine

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

Results 15 hour absorbance minus initial absorbance with 1 mM TheophyllineNO TETRACYCLINE LOW TETRACYCLINE HIGH TETRACYCLINE

J119140+J119303(pT5-TheoRiboSwitch-TetA + high promoter-high c. dog-eCDM8)

0.179 0.162 0.636

J119140+J119304(pT5-TheoRiboSwitch-TetA + low promoter-low c. dog-eCDM8)

0.288 0.246 0.170

J11940 in pSB4c5(pT5-TheoRiboSwitch-TetA)

0.263 n/a 0.193

J11940 in pSB1a2(pT5-TheoRiboSwitch-TetA)

0.278 n/a 0.214

K315009(pLacI-RBS-RFP-RBS-TetA)

0.325 n/a 0.120

J100111(pT5-RBS-CDM8-pT5-TheoRiboswitch-TetA)

0.560 0.523 -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.

Results 15 hour absorbance minus initial absorbance with 5 mM Caffeine

NO TETRACYCLINE LOW TETRACYCLINE HIGH TETRACYCLINE

J119140+J119303(pT5-TheoRiboSwitch-TetA + high promoter-high c. dog-eCDM8)

0.012 0 -0.004

J119140+J119304(pT5-TheoRiboSwitch-TetA + low promoter-low c. dog-eCDM8)

0.019 0 -0.006

J11940 in pSB4c5(pT5-TheoRiboSwitch-TetA)

0.173 n/a 0.008

J11940 in pSB1a2(pT5-TheoRiboSwitch-TetA)

0.181 n/a 0.006

K315009(pLacI-RBS-RFP-RBS-TetA)

0.179 n/a 0.021

J100111(pT5-RBS-CDM8-pT5-TheoRiboswitch-TetA)

0.304 -0.002 -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.