Part:BBa_K1529797:Experience

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

Result2 3OC12HSL-dependent C4HSL production assay

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

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