August/4 October 2009

From 2009.igem.org

1.sencivity test We conducted a preliminary test on one of our receiver circuits, the LuxR receiver. A GFP coding region was attached behind the receiver's pLux promoter and the construct designated X1. This was tested with AHL at 4 different concentrations: 0, 10nM, 1uM, 100uM. In addition, we also included a pLux-GFP (no LuxR coding region) circuit and a pTet-GFP (constitutive ON) circuit as 'negative' and 'positive' controls respectively.

Pre-incubated cells were inoculated into 20ml LB-Amp medium and 1ml was taken out of each incubation flask, washed and resuspended in MiliQ water and fluorescence measured (excitation 488nm, emission 508nm) every hour. The experiment's results more or less agreed with a previous test on the similarly-constructed BBa_T9002, in that maximum fluorescence was obtained for AHL concentrations of around 10-20nM.

One problem that occurred to us halfway through the experiment was that since GFP fluorescence level probably correlates linearly with cell density, so we should have taken an OD600 (optical density at 600nm) reading with each fluorescence sampling, in order to measure actual per-cell GFP expression. This experiment will be repeated next week along with other signaling systems when their respective AHLs arrive.

The pTet-GFP circuit which was supposed to constitutively express GFP, was found to have a very low level of fluorescence. The part was judged faulty and discarded (a new, properly fluorescing GFP part was constructed in its stead).

Another unexpected result was that the fluorescence for the 'negative control', pLux-GFP was found to be the highest. 2 things can be inferred: that 1) some leaky expression occurs at the pLux promoter *even in the absence of LuxR protein*, and 2) higher cell concentration, perhaps due to the absence of AHL or LuxR, resulted in a high overall GFP expression.

2.colony check

sample no,     no. of colony
   58                         ++
   59                           +
   60                         ++
   61                         ++
   62                       +++
   63                          ++ 
   64                        +++
   65                           +

3.digaiton

K204067

Vector		Insert
1-23L	6	2-16L	10
EcoRI	1	EcoRI	1
XbaI	1	SpeI	1
No.2	2	No.2	2
dH₂O	10	dH₂O	6
total	20uL	total	20uL

K204068

Vector		Insert
1-23L	10	1-19B	5
EcoRI	1	EcoRI	1
XbaI	1	SpeI	1
No.2	2	No.2	2
dH₂O	6	dH₂O	11
total	20uL	total	20uL

↓
37°C, 2hr
gel cut & purification
ligation 3hr

4.min prep

sample       conc.
  2-18F       213.7 ng/uL

5.transformation

sample no.
 58 , 59 , 60 , 61 , 62 , 63 , 64 , 65

6.color intensity check

color parts check

7.digation

K204064

Vector		Insert
2-16N	3	2-18H	6
EcoRI	1	EcoRI	1
XbaI	1	SpeI	1
No.2	2	No.2	2
dH₂O	13	dH₂O	10
total	20uL	total	20uL

K204065

Vector		Insert
2-16L	3	2-18F	10
SpeI	1	XbaI	1
PstI	1	PstI	1
No.2	2	No.2	2
dH₂O	13	dH₂O	6
total	20uL	total	20uL

↓
37°C, 2hr
gel cut & purification
ligation O/N

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