Difference between revisions of "Part:BBa K1529797:Experience"
Line 1: Line 1:
 
<partinfo>BBa_K1529265 short</partinfo>
 
<partinfo>BBa_K1529265 short</partinfo>
 
__TOC__
 
__TOC__
−
We confirmed whether the expression of CmR and C4HSL depends on the induction of 3OC12HSL.  We inserted <i>lux</i> promoter (activated by 3OC12HSL-LuxR complex) upstream of <i>cmR</i> and <i>rhlI</i>, as an inducible promoter.<br>
 
  
−
[[Image:Assay1_Flowchart.png|thumb|center|300px|<b>Fig. 1.</b> Flow chart of Assay1.]]
 
−
[[Image:Assay2_Flowchart.png|thumb|center|300px|<b>Fig. 2.</b> Flow chart of Assay2.]]
 
  
−
アッセイの概要
+
==Materials and Methods==
−
 
+
−
==3OC12HSL-dependent CmR expression==
+
−
 
+
−
===3OC12HSL-dependent CmR expression Result===
+
−
<b>1. Plasmid construction</b><br>
+
−
[[Image:Plasmid construction for assay1.png|thumb|right|300px|<b>Fig. 2.</b> Plasmid construction for assay1]]
+
−
Sample:<br>
+
−
pSB3K3-Plux-<i>cmR</i>-<i>rhlI</i> (<partinfo>BBa_K1529265</partinfo>)<br>
+
−
pSB6A1-Ptet-<i>luxR</i>-Plac-<i>rfp</i><br>
+
−
 
+
−
Positive control:<br>
+
−
pSB3K3-PlacIq-<i>cmR</i><br>
+
−
pSB6A1-Ptet-<i>luxR</i>-Plac-<i>rfp</i><br>
+
−
 
+
−
Negative control:<br>
+
−
pSB3K3-(Promoter less)-<i>cmR</i><br>
+
−
pSB6A1-Ptet-<i>luxR</i>-Plac-<i>rfp</i><br>
+
−
 
+
−
 
+
−
<b>2. Assay protocol</b><br>
+
−
*2-0 Strains<br>
+
−
DH5alpha (<i>E. coli</i> of high competence)<br>
+
−
JM2.300 (lacI22 <i>E. coli</i>)<br>
+
−
 
+
−
*2-1 Media<br>
+
−
Mix everything together in 1,000 mL autoclaved Elix H<sub>2</sub>O<br>
+
−
LB
+
−
{| class="wikitable" cellpadding="6"
+
−
|Bacto tryptone||10 g/L
+
−
|-
+
−
|Yeast Extract||&nbsp;&nbsp;5 g/L
+
−
|-
+
−
|NaCl||10 g/L
+
−
|}
+
−
 
+
−
*2-2 Others<br>
+
−
[ Antibiotics ]<br>
+
−
Ampisillin, Kanamycin, Chloramphenicol<br>
+
−
[ Inducer ]<br>
+
−
3OC12HSL dissolved in DMSO (>100 µM)<br>
+
−
 
+
−
*2-3 Protocol<br>
+
−
[ Preparation ]<br>
+
−
1.Transform JM2.300 with pSB3K3-Plux-M13-Plac-<i>GFP</i> and pSB6A1-Ptet-<i>luxR</i> <br>
+
−
2.Grow overnight culture (Amp + Kan) of the transformed DH5alpha and JM109 at 37°C.<br>
+
−
3.Take 30 µL of the overnight culture into 3 mL LB (Amp + Kan).  (=> fresh culture)<br>
+
−
4.Incubate the fresh culture at 37°C for 2 hours.<br>
+
−
5.Add 3 µL of 5 µM 3OC6HSL in DMSO (final concentration: 5 nM) to the fresh culture and incubate at 37°C for 4 hours.<br>
+
−
6.Spin the overnight culture of the transformed DH5alpha at 9,000g for 1 minute.<br>
+
−
7.Pipette the supernatant into a 1.5 mL tube.<br>
+
−
8.Dilute it 100 times with water. (=> phage-particle-solution)<br>
+
−
 
+
−
[ Plaque formation ]<br>
+
−
9.Transform JM109 with pSB6A1-Ptet-<i>luxR</i> <br>
+
−
10.Grow overnight culture of the transformed JM109 at 37°C.<br>
+
−
11.Melt YT soft agar using a microwave.<br>
+
−
12.Add ampicillin to the YT soft agar.<br>
+
−
13.Dispense 3.5 mL of the melted soft agar to a new round tube, and keep them at 50°C in an incubator.<br>
+
−
14.Dispense 400 µL of overnight culture of the transformed JM109 to a 1.5 mL tube.<br>
+
−
15.Into the 1.5 mL tube, add 100 µL of the phage-particle-solution.<br>
+
−
16.Transfer all the content of the 1.5 mL tube to the dispensed soft agar, mix them, and decant all of it on a YT plate.<br>
+
−
17.Wait for YT soft agar to solidify at room temperature (for about 5 minutes).<br>
+
−
18.Put an autoclaved piece of filter paper on the plate, and drip 20 mL of 3OC6HSL in DMSO (100 µM, 5 µM or DMSO only) on the piece of filter paper.<br>
+
  
 
===3OC12HSL-dependent CmR expression Protocol===
 
===3OC12HSL-dependent CmR expression Protocol===
−
<b>1-1. Plasmid construction</b><br>
 
−
pSB3K3-Plux-<i>rmR<i/>-<i>rhlI</i> (<partinfo>BBa_K1529265</partinfo>)<br>
 
−
pSB6A1-Ptet-<i>luxR</i>-Plac-<i>rfp</i><br>
 
  
−
[[Image:titech2013_parts_K1139021_exp_Fig2.jpg|thumb|center|300px|<b>Fig. 2.</b> Plasmid construction for assay]]
 
  
−
<b>1-2. Assay protocol</b><br>
+
===3OC12HSL-dependent C4HSL production Protocol===
−
*2-0 Strains<br>
+
−
JM2.300 <br>
+
  
−
*2-1 Media<br>
 
−
Mix everything together in 1,000 mL autoclaved Elix H<sub>2</sub>O<br>
 
−
LB
 
−
{| class="wikitable" cellpadding="6"
 
−
|Bacto tryptone||10 g/L
 
−
|-
 
−
|Yeast Extract||&nbsp;&nbsp;5 g/L
 
−
|-
 
−
|NaCl||10 g/L
 
−
|}
 
  
−
*2-2 Others<br>
+
==Results==
−
3OC12HSL dissolved in DMSO (>100 µM)<br>
+
−
Autoclaved pieces of filter paper (about 1.5 cm in diameter) <br>
+
  
−
*2-3 Protocol<br>
+
===3OC12HSL-dependent CmR expression Result===
−
[ Preparation ]<br>
+
−
1.Transform DH5alpha with pSB3K3-Plux-M13-Plac-<i>GFP</i> and pSB6A1-Ptet-<i>luxR</i> <br>
+
−
2.Grow overnight culture (Amp + Kan) of the transformed DH5alpha and JM109 at 37°C.<br>
+
−
3.Take 30 µL of the overnight culture into 3 mL LB (Amp + Kan).  (=> fresh culture)<br>
+
−
4.Incubate the fresh culture at 37°C for 2 hours.<br>
+
−
5.Add 3 µL of 5 µM 3OC6HSL in DMSO (final concentration: 5 nM) to the fresh culture and incubate at 37°C for 4 hours.<br>
+
−
6.Spin the overnight culture of the transformed DH5alpha at 9,000g for 1 minute.<br>
+
−
7.Pipette the supernatant into a 1.5 mL tube.<br>
+
−
8.Dilute it 100 times with water. (=> phage-particle-solution)<br>
+
−
 
+
−
[ Plaque formation ]<br>
+
−
9.Transform JM109 with pSB6A1-Ptet-<i>luxR</i> <br>
+
−
10.Grow overnight culture of the transformed JM109 at 37°C.<br>
+
−
11.Melt YT soft agar using a microwave.<br>
+
−
12.Add ampicillin to the YT soft agar.<br>
+
−
13.Dispense 3.5 mL of the melted soft agar to a new round tube, and keep them at 50°C in an incubator.<br>
+
−
14.Dispense 400 µL of overnight culture of the transformed JM109 to a 1.5 mL tube.<br>
+
−
15.Into the 1.5 mL tube, add 100 µL of the phage-particle-solution.<br>
+
−
16.Transfer all the content of the 1.5 mL tube to the dispensed soft agar, mix them, and decant all of it on a YT plate.<br>
+
−
17.Wait for YT soft agar to solidify at room temperature (for about 5 minutes).<br>
+
−
18.Put an autoclaved piece of filter paper on the plate, and drip 20 mL of 3OC6HSL in DMSO (100 µM, 5 µM or DMSO only) on the piece of filter paper.<br>
+
−
 
+
−
==3OC12HSL-dependent C4HSL expression==
+
−
===3OC12HSL-dependent C4HSL expression===
+
  
  
  
−
===3OC12HSL-dependent C4HSL expression===
+
===3OC12HSL-dependent C4HSL production Result===
  
  
−
For more information, see [http://2014.igem.org/Team:Tokyo_Tech our work in Tokyo_Tech 2013 wiki].
+
For more information, see [http://2014.igem.org/Team:Tokyo_Tech/Experiment/3OC12HSL-dependent_C4HSL_production our work in Tokyo_Tech 2014 wiki].
  
−
==Applications of BBa_K1529265==
+
==Applications of BBa_K1529797==
  
 
==User Reviews==
 
==User Reviews==
−
<!-- DON'T DELETE --><partinfo>BBa_K1529265 StartReviews</partinfo>
+
<!-- DON'T DELETE --><partinfo>BBa_K1529797 StartReviews</partinfo>
 
<!-- Template for a user review
 
<!-- Template for a user review
 
{|width='80%' style='border:1px solid gray'
 
{|width='80%' style='border:1px solid gray'
 
|-
 
|-
 
|width='10%'|
 
|width='10%'|
−
<partinfo>BBa_K1529265 AddReview number</partinfo>
+
<partinfo>BBa_K1529797 AddReview number</partinfo>
 
<I>Username</I>
 
<I>Username</I>
 
|width='60%' valign='top'|
 
|width='60%' valign='top'|
Line 144: Line 36:
 
|};
 
|};
 
<!-- End of the user review template -->
 
<!-- End of the user review template -->
−
<!-- DON'T DELETE --><partinfo>BBa_K1529265 EndReviews</partinfo>
+
<!-- DON'T DELETE --><partinfo>BBa_K1529797 EndReviews</partinfo>

Revision as of 15:49, 19 October 2014

Plux_CmR_RhlI


Materials and Methods

3OC12HSL-dependent CmR expression Protocol

3OC12HSL-dependent C4HSL production Protocol

Results

3OC12HSL-dependent CmR expression Result

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].

Applications of BBa_K1529797

User Reviews

UNIQ58ce4bb04c426cce-partinfo-00000001-QINU UNIQ58ce4bb04c426cce-partinfo-00000002-QINU