August/4 October 2009

From 2009.igem.org

(Difference between revisions)
(New page: 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. Th...)
 
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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.
+
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.<br>
 +
<br>
 +
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.<br>
-
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.
+
<br>
 +
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.<br>
 +
<br>
 +
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).<br>
 +
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.<br>
 +
<br>
 +
2.colony check<br>
 +
sample no,    no. of colony
 +
    58                        ++
 +
    59                          +
 +
    60                        ++
 +
    61                        ++
 +
    62                      +++
 +
    63                          ++
 +
    64                        +++
 +
    65                          +
-
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.
+
3.digaiton<br>
-
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).
+
K204067
 +
<table border="1" Frame="box">
 +
<tr><th colspan="2">Vector</th><th colspan="2">Insert</th></tr>
 +
<tr><td>1-23L</td><td>6</td><td>2-16L</td><td>10</td></tr>
 +
<tr><td>EcoRI</td><td>1</td><td>EcoRI</td><td>1</td><tr>
 +
<tr><td>XbaI</td><td>1</td><td>SpeI</td><td>1</td><tr>
 +
<tr><td> No.2</td><td>2</td><td>No.2</td><td>2</td></tr>
 +
<tr><td>dH<sub>2</sub>O</td><td>10</td><td>dH<sub>2</sub>O</td><td>6</td></tr>
 +
<tr><td>total</td><td>20uL</td><td>total</td><td>20uL</td></tr>
 +
</table>
 +
<br>
-
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.
+
 
 +
K204068
 +
<table border="1" Frame="box">
 +
<tr><th colspan="2">Vector</th><th colspan="2">Insert</th></tr>
 +
<tr><td>1-23L</td><td>10</td><td>1-19B</td><td>5</td></tr>
 +
<tr><td>EcoRI</td><td>1</td><td>EcoRI</td><td>1</td><tr>
 +
<tr><td>XbaI</td><td>1</td><td>SpeI</td><td>1</td><tr>
 +
<tr><td> No.2</td><td>2</td><td>No.2</td><td>2</td></tr>
 +
<tr><td>dH<sub>2</sub>O</td><td>6</td><td>dH<sub>2</sub>O</td><td>11</td></tr>
 +
<tr><td>total</td><td>20uL</td><td>total</td><td>20uL</td></tr>
 +
</table>
 +
<br>
 +
↓<br>
 +
37°C, 2hr<br>
 +
gel cut & purification<br>
 +
ligation 3hr<br>
 +
 
 +
<br>
 +
4.min prep
 +
sample      conc.
 +
  2-18F      213.7 ng/uL
 +
 
 +
<br>
 +
5.transformation<br>
 +
sample no.
 +
  58 , 59 , 60 , 61 , 62 , 63 , 64 , 65
 +
 
 +
<br>
 +
6.color intensity check<br>
 +
color parts check
 +
 
 +
<br>
 +
7.digation<br>
 +
 
 +
K204064
 +
<table border="1" Frame="box">
 +
<tr><th colspan="2">Vector</th><th colspan="2">Insert</th></tr>
 +
<tr><td>2-16N</td><td>3</td><td>2-18H</td><td>6</td></tr>
 +
<tr><td>EcoRI</td><td>1</td><td>EcoRI</td><td>1</td><tr>
 +
<tr><td>XbaI</td><td>1</td><td>SpeI</td><td>1</td><tr>
 +
<tr><td> No.2</td><td>2</td><td>No.2</td><td>2</td></tr>
 +
<tr><td>dH<sub>2</sub>O</td><td>13</td><td>dH<sub>2</sub>O</td><td>10</td></tr>
 +
<tr><td>total</td><td>20uL</td><td>total</td><td>20uL</td></tr>
 +
</table>
 +
<br>
 +
 
 +
 
 +
 
 +
K204065
 +
<table border="1" Frame="box">
 +
<tr><th colspan="2">Vector</th><th colspan="2">Insert</th></tr>
 +
<tr><td>2-16L</td><td>3</td><td>2-18F</td><td>10</td></tr>
 +
<tr><td>SpeI</td><td>1</td><td>XbaI</td><td>1</td><tr>
 +
<tr><td>PstI</td><td>1</td><td>PstI</td><td>1</td><tr>
 +
<tr><td> No.2 </td><td>2</td><td>No.2</td><td>2</td></tr>
 +
<tr><td>dH<sub>2</sub>O</td><td>13</td><td>dH<sub>2</sub>O</td><td>6</td></tr>
 +
<tr><td>total</td><td>20uL</td><td>total</td><td>20uL</td></tr>
 +
</table>
 +
↓<br>
 +
37°C, 2hr<br>
 +
gel cut & purification<br>
 +
ligation O/N<br>
 +
 
 +
 
 +
[https://2009.igem.org/Team:Osaka/NOTES back to NOTES]

Latest revision as of 00:48, 21 October 2009

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

VectorInsert
1-23L62-16L10
EcoRI1EcoRI1
XbaI1SpeI1
No.22No.22
dH2O10dH2O6
total20uLtotal20uL



K204068

VectorInsert
1-23L101-19B5
EcoRI1EcoRI1
XbaI1SpeI1
No.22No.22
dH2O6dH2O11
total20uLtotal20uL


↓
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

VectorInsert
2-16N32-18H6
EcoRI1EcoRI1
XbaI1SpeI1
No.22No.22
dH2O13dH2O10
total20uLtotal20uL



K204065

VectorInsert
2-16L32-18F10
SpeI1XbaI1
PstI1PstI1
No.2 2No.22
dH2O13dH2O6
total20uLtotal20uL

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


back to NOTES

Retrieved from "http://2009.igem.org/August/4_October_2009"