Difference between revisions of "Part:BBa K1529797:Experience"
Line 4: Line 4:
  
 
==Materials and Methods==
 
==Materials and Methods==
 +
-Strain<br>
 +
All the samples were JM2.300 strain.
  
 
===3OC12HSL-dependent CmR expression Protocol===
 
===3OC12HSL-dependent CmR expression Protocol===
 +
<b>1.Construction</b><br>
 +
A. Ptet-LuxR-Plac-RFP(pSB6A1), Plux-CmR-RhlI(pSB3K3)<br>
  
 +
B. Ptet-GFP-Ptet-RhlR (psB6A1), PlacIq-CmR (pSB3K3) (Positive control)<br>
  
−
===3OC12HSL-dependent C4HSL production Protocol===
 
  
 +
<b>2.Assay protocol</b><br>
 +
1.Prepare the overnight culture of cell A and B at 37°C.<br>
 +
2.Make a 1:100 dilution in 3 mL of fresh LB containing antibiotics and grow the cell at 37°C until the observed OD590 reaches 0.5 (→fresh culture)<br>
 +
3. Add 30 microL of suspension in the following medium.<br>
 +
　　　1) 3 mL of LB containing Amp and Kan + 30 microL C4HSL (final concentration is 500 microM)<br>
 +
　　　2) 3 mL of LB containing Amp and Kan + 30 microL DMSO<br>
 +
　　　3) 3 mL of LB containing Amp, Kan and Cm (final concentration is 100microg / mL) + 30 microL C4HSL (final concentration is 500 microM)<br>
 +
　　　4) 3 mL of LB containing Amp, Kan and Cm (final concentration is 100microg / mL) + 30 microL DMSO<br>
 +
4. Grow the samples of sender cells at 37°C for more than 10 hours. Measure optical density every hour. (If optical density is over 1.0, dilute the cell medium.)<br>
  
−
==Results==
 
  
−
===3OC12HSL-dependent CmR expression Result===
+
===3OC12HSL-dependent C4HSL production Protocol===
 +
<b>1.Construction</b><br>
 +
<strong>Sender</strong><br>
 +
A. Ptet-LuxR-Plac-RFP(pSB6A1), Plux-CmR-RhlI(pSB3K3)<br>
 +
B. Ptet-LuxR-Plac-RFP(pSB6A1), Plux-CmR(pSB3K3)...Negative control<br>
 +
<strong>Reporter</strong><br>
 +
C. Ptet-RhlR(pSB6A1), Plux-GFP(pSB3K3)<br>
 +
D. Ptet-RhlR(pSB6A1), PlacUV5-GFP(pSB3K3)...Positive control<br>
 +
E. Ptet-RhlR(pSB6A1),　Promoter-less-GFP(pSB3K3)...Negative control<br>
  
  
 +
<b>2.Assay protocol</b><br>
 +
==Results==
  
 +
===3OC12HSL-dependent CmR expression Result===
 +
We tested two types of culture condition which contains different concentration of chloramphenicol(Cm). (0 and 100 microg / mL)<br>
 +
Fig. 3-4-3-1 and Fig. 3-4-3-2 show the condition in the absence and the presence of chloramphenicol, respectively.<br>
 +
<br>
 +
Fig. 3-4-3-1 shows that every cell can grow in the absence of chloramphenicol.
 +
[[Image:3OC12HSL-dependent_CmR_expression_Result_Cm+.png|thumb|left|600px|<b>Fig. 1. </b>Plasmids]]<br>
 +
[[Image:3OC12HSL-dependent_CmR_expression_Result_Cm-.png|thumb|right|600px|<b>Fig. 2. </b>Plasmids]]<br>
 +
<br>
 +
On the other hand, in the presence of chloramphenicol, the cell containing Plux-CmR-RhlI can grow only when it was induced by 3OC12HSL.<br>
 +
Without the induction of 3OC12HSL, the cell cannot express CmR and cannot grow in the presence of chloramphenicol.<br>
 +
As a result, we confirmed that Plux-CmR-RhlI expressed CmR when induced by 3OC12HSL as expected.
 
===3OC12HSL-dependent C4HSL production Result===
 
===3OC12HSL-dependent C4HSL production Result===
  

Revision as of 16:09, 19 October 2014

Plux_CmR_RhlI


Materials and Methods

-Strain
All the samples were JM2.300 strain.

3OC12HSL-dependent CmR expression Protocol

1.Construction
A. Ptet-LuxR-Plac-RFP(pSB6A1), Plux-CmR-RhlI(pSB3K3)

B. Ptet-GFP-Ptet-RhlR (psB6A1), PlacIq-CmR (pSB3K3) (Positive control)


2.Assay protocol
1.Prepare the overnight culture of cell A and B at 37°C.
2.Make a 1:100 dilution in 3 mL of fresh LB containing antibiotics and grow the cell at 37°C until the observed OD590 reaches 0.5 (→fresh culture)
3. Add 30 microL of suspension in the following medium.
　　　1) 3 mL of LB containing Amp and Kan + 30 microL C4HSL (final concentration is 500 microM)
　　　2) 3 mL of LB containing Amp and Kan + 30 microL DMSO
　　　3) 3 mL of LB containing Amp, Kan and Cm (final concentration is 100microg / mL) + 30 microL C4HSL (final concentration is 500 microM)
　　　4) 3 mL of LB containing Amp, Kan and Cm (final concentration is 100microg / mL) + 30 microL DMSO
4. Grow the samples of sender cells at 37°C for more than 10 hours. Measure optical density every hour. (If optical density is over 1.0, dilute the cell medium.)


3OC12HSL-dependent C4HSL production Protocol

1.Construction
Sender
A. Ptet-LuxR-Plac-RFP(pSB6A1), Plux-CmR-RhlI(pSB3K3)
B. Ptet-LuxR-Plac-RFP(pSB6A1), Plux-CmR(pSB3K3)...Negative control
Reporter
C. Ptet-RhlR(pSB6A1), Plux-GFP(pSB3K3)
D. Ptet-RhlR(pSB6A1), PlacUV5-GFP(pSB3K3)...Positive control
E. Ptet-RhlR(pSB6A1),　Promoter-less-GFP(pSB3K3)...Negative control


2.Assay protocol

Results

3OC12HSL-dependent CmR expression Result

We tested two types of culture condition which contains different concentration of chloramphenicol(Cm). (0 and 100 microg / mL)
Fig. 3-4-3-1 and Fig. 3-4-3-2 show the condition in the absence and the presence of chloramphenicol, respectively.

Fig. 3-4-3-1 shows that every cell can grow in the absence of chloramphenicol.

Fig. 1. Plasmids

Fig. 2. Plasmids


On the other hand, in the presence of chloramphenicol, the cell containing Plux-CmR-RhlI can grow only when it was induced by 3OC12HSL.
Without the induction of 3OC12HSL, the cell cannot express CmR and cannot grow in the presence of chloramphenicol.
As a result, we confirmed that Plux-CmR-RhlI expressed CmR when induced by 3OC12HSL as expected.

3OC12HSL-dependent C4HSL production Result

For more information, see [http://2014.igem.org/Team:Tokyo_Tech/Experiment/3OC12HSL-dependent_C4HSL_production our work in Tokyo_Tech 2014 wiki].

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