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Part:BBa_K1139022:Experience

Designed by: Naoki Watarai   Group: iGEM13_Tokyo_Tech   (2013-09-24)

PlacIq-M13-Plac-GFP on pSB3

Materials and Methods

1. Plasmid construction
pSB3K3- PlacIq-M13-Plac-GFP (BBa_K1139020)
pSB3K3-M13-Plac-GFP (BBa_K1139022)

Fig. 1. Plasmid construction for assay






















2. Strain
DH5alpha (Ecoli of high competence)
JM109 (F+ strain Ecoli)

3. 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

4. Protocol

  • Preparation

1.Transform DH5alpha with pSB3K3- PlacIq-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.

Result

The result of the plaque forming assay is showed in Fig. 2. M13 phage genes and M13 origin worked together with lacIq 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- PlacIq-M13) at 9,000g. Mixture of 3.5 mL YT soft agar, 100 µL of X100 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 µL of overnight culture of JM109 was poured on a YT plate.

For more information, see [http://2013.igem.org/Team:Tokyo_Tech/Experiment/pSB-M13_Plasmid_Assay our work in Tokyo_Tech 2013 wiki].

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