Plaque Forming Assay PlacI^q

1. Introduction

We confirmed that M13 phage genes and M13 origin worked properly with pSB origin, using a plaque forming assay. The plasmid construction is as follows. First, from M13mp18 phage vector, we amplified the sequence that includes from g2p RBS to M13 origin (with prefix and suffix sequence), by PCR (Fig. 1). Second, we inserted the amplified sequence to pSB3K3 backbone. Finally, upstream of the RBS-g2p, we inserted PlacI^q (constitutive) promoter.

Fig. 1-1. Our designed plasmid constrction

2. Materials and Methods

2-1. Plasmid construction

pSB3K3-PlacI^q-M13-Plac-GFP (BBa_K1139020)

pSB3K3-M13-Plac-GFP (BBa_K113922)

2-2. Strains

DH5alpha (E. coli of high competence)

JM109 (F+ strain E. coli)

2-3. Media

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

2-4. Protocol

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 9,000 g for 1 min.

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 microL of overnight culture of JM109 to a 1.5 mL tube.

9.Into the 1.5 mL tube, add 100 microL 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.

3. Result

The result of the plaque forming assay is showed in Fig. 3-1. M13 phage genes and M13 origin worked together with lacI^q promoter and pSB origin.

Fig. 3-1A. 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 9,000 g. A mixture of 3.5 mL YT soft agar, 100 microL of 100X supernatant and 400 microL of overnight culture of JM109 was poured on a YT plate.

Fig. 3-1B. A mixture of only 3.5 mL YT soft agar and 500 microL of overnight culture of JM109 was poured on a YT plate.