Part:BBa_K1139020:Experience

BBa_K1139020

1. Materials and Method

1-0 Plasmid construction
pSB3K3- PlacI^q-M13-Plac-GFP (BBa_K1139020)
pSB3K3-M13-Plac-GFP (BBa_K1139022)

Fig. 1. Plasmid construction for assay

1-1 Strain
DH5alpha (Ecoli of high competence)
JM109 (F+ strain Ecoli)

1-2 Media
Mix everything together in 1000 mL autoclaved Elix H2O
LB

YT soft agar

YT plate

1-3Protocol

Preparation

1.Transform DH5alpha with pSB3K3- PlacI^q-M13.
2.Grow overnight culture of the transformed DH5alpha and JM109 at 37°C.

Plaque formation

3.Spin the overnight culture of the transformed DH5alpha at 9,000g for 1 minute.
4.Pipette the supernatant into a 1.5 mL tube.
5.Dilute it 100 times with water. (=>phage-particle-solution)
6.Melt YT soft agar using a microwave.
7.Dispense 3.5 mL of the melted soft agar to a new round tube, and keep them at 50°C in an incubator.
8.Dispense 400 µL of overnight culture of JM109 to a 1.5 mL tube.
9.Into the 1.5 mL tube, add 100 µL of the phage-particle-solution.
10.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.

2. Result

The result of the plaque forming assay is showed in Fig. 2. M13 phage genes and M13 origin worked together with lacI^q promoter and pSB origin.

Fig. 2-A. The plaques

Fig. 2-B. The negative control

Fig. 2-A.
The plaques were formed using JM109 overnight culture and phage-particle-solution. The phage particles were obtained from the supernatant of the overnight culture of transformed DH5alpha (with pSB3K3- PlacI^q-M13) at 9,000g. Mixture of 3.5 mL YT soft agar, 100 µL of X 100 supernatant and 400 µL of overnight culture of JM109 was poured on a YT plate.

Fig. 2-B.
A mixture of only 3.5 mL YT soft agar and 500 microL of overnight culture of JM109 was poured on a YT plate.

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