Part:BBa_K1139021:Experience

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 3OC6HSL-LuxR 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.

Materials and Methods

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 1,000 mL autoclaved Elix H₂O
LB

Bacto tryptone	10 g/L
Yeast Extract	5 g/L
NaCl	10 g/L

YT plate

Bacto tryptone	8 g/L
Yeast Extract	5 g/L
NaCl	5 g/L
Agarose	15 g/L

YT soft agar

Bacto tryptone	8 g/L
Yeast Extract	5 g/L
NaCl	5 g/L
Agarose	6 g/L

• 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.

Result

The result of the plaque forming assay is showed in Fig. 3. On the plate, the plaques were formed at some distance from the piece of filter paper. The result shows that phage release is regulated by induction of 3OC6HSL. We also analyze the distribution of the plaques (Fig. 4). The histogram result implicates that the plaques are formed only at the appropriate concentration of 3OC6HSL in medium (Fig. 5). In the neighborhood of the piece of filter paper, the expression of g2p is so activated that the phage particles cannot be produced efficiently, because the production of the coat proteins largely exceeds that of the single stranded DNA, and vice versa.

Fig. 3. The plaques formed with 3OC6HSL induction (plaque plotted)

The plaques were formed using JM109 (with pSB6A1-Ptet-luxR) overnight culture as a lawn and phage-particle-solution. The phage particles were obtained from the supernatant of the fresh culture (+3OC6HSL) of transformed DH5alpha (with pSB3K3-Plux-M13) at 9,000g. A mixture of 3.5 mL YT soft agar, 100 µL of X 100 supernatant and 400 µL of overnight culture of JM109 (with pSB6A1-Ptet-luxR) was poured on a YT plate. After solidifying the soft agar, we put a piece of filter paper on the plate and dripped 3OC6HSL in DMSO (or DMSO only) on the piece of filter paper.

Fig. 4. The analysis of distribution of the plaques

We totalize the distances from the center of the piece of filter paper to each plaque, by using ImageJ.

Fig. 5. The distribution histogram of the plaques

This histogram shows distance from the piece of paper (+ inducer) in a horizontal axis, and shows number of plaques over distance squared, which means the rate of plaque formation, in a vertical axis. The bin range is 0.2 cm.

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

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