August/10 October 2009

Today we conducted AHL sensitivity tests on 3 of our receiver parts (LuxR, LasR, RhlR). Each part had a GFP coding region attached so that GFP expression would be triggered by the respective promoters (pLux, pLas, pRhl), and the constructs were designated X1, X2 and X3 respectively. These were then tested with 0, 10nM and 100nM AHL concentrations by incubation in LB medium at 30 degrees Celsius. We took 1ml of sample from each culture and measured its OD600 (optical density at 600nm), then washed the cells and resuspended them in MiliQ water and then measured their GFP fluorescence (excitation 455nm, emission 507nm). This was repeated once every hour to obtain a graph of fluorescence versus OD600. From our tests it appeared that the only part which showed an increased fluorescence/OD600 ratio in the presence of AHL was RhlR. This shall be incorporated into the final circuit hopefully in time for delivery to iGEM Headquarters. 2.min prep sapmle    conc. 21 m+       34 ng/uL 2-6O m+    45 20 m+        49 11 m+        28 48 m+        113 1-8I            19 75               104  74                166  71                69  32                23  68                62  68                72  67                102  67                64  33               37  1-8I             19 65               267

3.digation

K204058

K204062

K204073 ↓ 37 degree, 3hr gel cut & purification ligation

4.transformation sample 58, 62 , 73 , chiba parts(2 , 3, ,4, ) , 31 , 76 , 77 , 78

5.min prep sample           conc. chiba 1          169 ng/uL chiba 5           80 chiba 6           48 chiba 7           44 chiba 8           105 chiba 9           119

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