August/10 October 2009

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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

Vector		Insert
1-8E	2	1-8I	2
SpeI	1	XbaI	1
PstI	1	PstI	1
No.2	2	No.2	2
dH₂O	10	dH₂O	14
total	20uL	total	20uL

K204062

Vector		Insert
1-8E	4	33	4
SpeI	1	XbaI	1
PstI	1	PstI	1
No.2	2	No.2	2
dH₂O	12	dH₂O	12
total	20uL	total	20uL

K204073

Vector		Insert
1-8I	5	32	8
EcoRI	1	EcoRI	1
XbaI	1	SpeI	1
No.2	2	No.2	2
dH₂O	9	dH₂O	6
total	20uL	total	20uL

↓
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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