Difference between revisions of "Part:BBa K1529797:Experience"

 
Line 1: Line 1:
 +
<partinfo>BBa_K1529265 short</partinfo>
 +
__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>
  
−
__NOTOC__
+
[[Image:Assay1_Flowchart.png|thumb|center|300px|<b>Fig. 1.</b> Flow chart of Assay1.]]
−
This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
+
[[Image:Assay2_Flowchart.png|thumb|center|300px|<b>Fig. 2.</b> Flow chart of Assay2.]]
−
how you used this part and how it worked out.
+
  
−
===Applications of BBa_K1529797===
+
アッセイの概要
  
−
===User Reviews===
+
==Materials and Methods==
−
<!-- DON'T DELETE --><partinfo>BBa_K1529797 StartReviews</partinfo>
+
 
 +
===Assay1 3OC12HSL-dependent Plux-CmR-RhlI cell growth assay===
 +
<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>
 +
 
 +
===Assay2 3OC12HSL-dependent C4HSL production assay===
 +
<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>
 +
*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>
 +
3OC12HSL dissolved in DMSO (>100 µM)<br>
 +
Autoclaved pieces of filter paper (about 1.5 cm in diameter) <br>
 +
 
 +
*2-3 Protocol<br>
 +
[ 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>
 +
 
 +
==Result==
 +
===Result1 3OC12HSL-dependent Plux-CmR-RhlI cell growth assay===
 +
 
 +
 
 +
 
 +
===Result2 3OC12HSL-dependent C4HSL production assay===
 +
 
 +
 
 +
For more information, see [http://2014.igem.org/Team:Tokyo_Tech our work in Tokyo_Tech 2013 wiki].
 +
 
 +
==Applications of BBa_K1529265==
 +
 
 +
==User Reviews==
 +
<!-- DON'T DELETE --><partinfo>BBa_K1529265 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_K1529797 AddReview number</partinfo>
+
<partinfo>BBa_K1529265 AddReview number</partinfo>
 
<I>Username</I>
 
<I>Username</I>
 
|width='60%' valign='top'|
 
|width='60%' valign='top'|
Line 18: Line 144:
 
|};
 
|};
 
<!-- End of the user review template -->
 
<!-- End of the user review template -->
−
<!-- DON'T DELETE --><partinfo>BBa_K1529797 EndReviews</partinfo>
+
<!-- DON'T DELETE --><partinfo>BBa_K1529265 EndReviews</partinfo>

Revision as of 05:25, 8 October 2014

Plux_CmR_RhlI

We confirmed whether the expression of CmR and C4HSL depends on the induction of 3OC12HSL. We inserted lux promoter (activated by 3OC12HSL-LuxR complex) upstream of cmR and rhlI, as an inducible promoter.

File:Assay1 Flowchart.png
Fig. 1. Flow chart of Assay1.
File:Assay2 Flowchart.png
Fig. 2. Flow chart of Assay2.

アッセイの概要

Materials and Methods

Assay1 3OC12HSL-dependent Plux-CmR-RhlI cell growth assay

1. Plasmid construction

Fig. 2. Plasmid construction for assay1

Sample:
pSB3K3-Plux-cmR-rhlI (BBa_K1529265)
pSB6A1-Ptet-luxR-Plac-rfp

Positive control:
pSB3K3-PlacIq-cmR
pSB6A1-Ptet-luxR-Plac-rfp

Negative control:
pSB3K3-(Promoter less)-cmR
pSB6A1-Ptet-luxR-Plac-rfp


2. Assay protocol

  • 2-0 Strains

DH5alpha (E. coli of high competence)
JM2.300 (lacI22 E. coli)

  • 2-1 Media

Mix everything together in 1,000 mL autoclaved Elix H2O
LB

Bacto tryptone 10 g/L
Yeast Extract   5 g/L
NaCl 10 g/L
  • 2-2 Others

[ Antibiotics ]
Ampisillin, Kanamycin, Chloramphenicol
[ Inducer ]
3OC12HSL dissolved in DMSO (>100 µM)

  • 2-3 Protocol

[ Preparation ]
1.Transform JM2.300 with pSB3K3-Plux-M13-Plac-GFP and pSB6A1-Ptet-luxR
2.Grow overnight culture (Amp + Kan) of the transformed DH5alpha and JM109 at 37°C.
3.Take 30 µL of the overnight culture into 3 mL LB (Amp + Kan). (=> fresh culture)
4.Incubate the fresh culture at 37°C for 2 hours.
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.
6.Spin the overnight culture of the transformed DH5alpha at 9,000g for 1 minute.
7.Pipette the supernatant into a 1.5 mL tube.
8.Dilute it 100 times with water. (=> phage-particle-solution)

[ Plaque formation ]
9.Transform JM109 with pSB6A1-Ptet-luxR
10.Grow overnight culture of the transformed JM109 at 37°C.
11.Melt YT soft agar using a microwave.
12.Add ampicillin to the YT soft agar.
13.Dispense 3.5 mL of the melted soft agar to a new round tube, and keep them at 50°C in an incubator.
14.Dispense 400 µL of overnight culture of the transformed JM109 to a 1.5 mL tube.
15.Into the 1.5 mL tube, add 100 µL of the phage-particle-solution.
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.
17.Wait for YT soft agar to solidify at room temperature (for about 5 minutes).
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.

Assay2 3OC12HSL-dependent C4HSL production assay

1-1. Plasmid construction
pSB3K3-Plux-rmR<i/>-<i>rhlI (BBa_K1529265)
pSB6A1-Ptet-luxR-Plac-rfp

Fig. 2. Plasmid construction for assay

1-2. Assay protocol

  • 2-0 Strains

JM2.300

  • 2-1 Media

Mix everything together in 1,000 mL autoclaved Elix H2O
LB

Bacto tryptone 10 g/L
Yeast Extract   5 g/L
NaCl 10 g/L
  • 2-2 Others

3OC12HSL dissolved in DMSO (>100 µM)
Autoclaved pieces of filter paper (about 1.5 cm in diameter)

  • 2-3 Protocol

[ Preparation ]
1.Transform DH5alpha with pSB3K3-Plux-M13-Plac-GFP and pSB6A1-Ptet-luxR
2.Grow overnight culture (Amp + Kan) of the transformed DH5alpha and JM109 at 37°C.
3.Take 30 µL of the overnight culture into 3 mL LB (Amp + Kan). (=> fresh culture)
4.Incubate the fresh culture at 37°C for 2 hours.
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.
6.Spin the overnight culture of the transformed DH5alpha at 9,000g for 1 minute.
7.Pipette the supernatant into a 1.5 mL tube.
8.Dilute it 100 times with water. (=> phage-particle-solution)

[ Plaque formation ]
9.Transform JM109 with pSB6A1-Ptet-luxR
10.Grow overnight culture of the transformed JM109 at 37°C.
11.Melt YT soft agar using a microwave.
12.Add ampicillin to the YT soft agar.
13.Dispense 3.5 mL of the melted soft agar to a new round tube, and keep them at 50°C in an incubator.
14.Dispense 400 µL of overnight culture of the transformed JM109 to a 1.5 mL tube.
15.Into the 1.5 mL tube, add 100 µL of the phage-particle-solution.
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.
17.Wait for YT soft agar to solidify at room temperature (for about 5 minutes).
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.

Result

Result1 3OC12HSL-dependent Plux-CmR-RhlI cell growth assay

Result2 3OC12HSL-dependent C4HSL production assay

For more information, see [http://2014.igem.org/Team:Tokyo_Tech our work in Tokyo_Tech 2013 wiki].

Applications of BBa_K1529265

User Reviews

UNIQ7d45e7e494323d83-partinfo-00000003-QINU UNIQ7d45e7e494323d83-partinfo-00000004-QINU