Difference between revisions of "Part:BBa K1139021:Experience"

Revision as of 10:19, 26 September 2013

Plux-M13-Plac-GFP on pSB3

We confirmed whether the release of M13 phage particle depends on the induction of g2p expression. We inserted lux promoter (activated by LuxR-3OC6HSL complex) upstream of g2p, as an inducible promoter.

Fig. 1. Flow chart of this experiment.

Prepare E. coli surrounded by a red circle. We add inducer (AHL) to the E. coli. Phage is released by induction of AHL. Spin the culture of the E. coli. Decant the supernatant of the culture including phages to soft agar ( the agar which is easy to melt ). Add lawn (E. coli containing pSB6A1-Ptet-luxR ) to the soft agar. Mix them in soft agar. Then, decant the soft agar to YT plate.

1. Materials and Method

1. Plasmid construction
pSB3K3-Plux-M13-Plac-GFP (BBa_K1139021)
pSB6A1-Ptet-luxR

Fig. 2. Plasmid construction for assay

2. Assay protocol

• 2-0 Strains
  

DH5alpha (E. coli of high competence)
JM109 (F+ strain E. coli)

• 2-1 Media
  

Mix everything together in 1000 mL autoclaved Elix H2O
LB

YT soft agar

YT plate

• 2-2 Others
  

3OC6HSL 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.

2. Result

Applications of BBa_K1139021

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