Team:Tokyo Tech/Experiment/Inducible Plaque Forming Assay

From 2013.igem.org

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<p>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 <i>g2p</i> 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-<i>g2p</i>, we inserted PlacI<sup>q</sup> (constitutive) promoter.  
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<p>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 <i>g2p</i> 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-<i>g2p</i>, we inserted PlacI<sup>q</sup> (constitutive) promoter.  
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<p>Yeast Extract  5 g/L
<p>Yeast Extract  5 g/L
</p>
</p>
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<p>NaCl:           10 g/L
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<p>NaCl          10 g/L
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</p><br>
<p>YT plate
<p>YT plate
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<p>Yeast Extract  5 g/L
<p>Yeast Extract  5 g/L
</p>
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<p>NaCl:           5 g/L
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<p>NaCl           5 g/L
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</p>
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<p>Agarose        15 g/L
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</p><br>
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<p>YT soft agar
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</p>
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<p>Bacto tryptone  8 g/L
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</p>
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<p>Yeast Extract  5 g/L
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</p>
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<p>NaCl           5 g/L
</p>
</p>
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<p>Agarose:       15 g/L
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<p>Agarose        6 g/L
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2-2-3Protocol
2-2-3Protocol
<p>Preparation
<p>Preparation
</p>
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1.Transform DH5alpha with pSB3K3-PlacI<sup>q</sup>-M13.
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<p>1.Transform DH5alpha with pSB3K3-PlacI<sup>q</sup>-M13.</p>
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2.Grow overnight culture of the transformed DH5alpha and JM109 at 37°C.
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<p>2.Grow overnight culture of the transformed DH5alpha and JM109 at 37°C.</p>
<p>Plaque formation
<p>Plaque formation
</p>
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3.Spin the overnight culture of the transformed DH5alpha at 9,000 g for 1 minute.
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<p>3.Spin the overnight culture of the transformed DH5alpha at 9,000 g for 1 minute.</p>
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4.Pipette the supernatant into a 1.5 mL tube.
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<p>4.Pipette the supernatant into a 1.5 mL tube.</p>
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5.Dilute it 100 times with water.  (→ phage-particle-solution)
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<p>5.Dilute it 100 times with water.  (-> phage-particle-solution)</p>
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6.Melt YT soft agar using a microwave.  
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<p>6.Melt YT soft agar using a microwave. </p>
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7.Dispense 3.5 mL of the melted soft agar to a new round tube, and keep them at 50°C in an incubator.
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<p>7.Dispense 3.5 mL of the melted soft agar to a new round tube, and keep them at 50°C in an incubator.</p>
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8.Dispense 400 microL of overnight culture of JM109 to a 1.5 mL tube.
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<p>8.Dispense 400 microL of overnight culture of JM109 to a 1.5 mL tube.</p>
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9.Into the 1.5 mL tube, add 100 microL of the phage-particle-solution.
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<p>9.Into the 1.5 mL tube, add 100 microL of the phage-particle-solution.</p>
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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.
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<p>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.</p>
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<p>The result of the plaque forming assay is showed in Fig 2  M13 phage genes and M13 origin worked together with lacI<sup>q</sup> promoter and pSB origin.
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<p>The result of the plaque forming assay is showed in Fig. 2. M13 phage genes and M13 origin worked together with <i>lacI<sup>q</sup></i> promoter and pSB origin.
</p>
</p>
<p>(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<sup>q</sup>-M13) at 9,000 g.  Mixture of 3.5 mL YT soft agar, 100 microL of X 100 supernatant and 400 microL of overnight culture of JM109 was poured on a YT plate.
<p>(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<sup>q</sup>-M13) at 9,000 g.  Mixture of 3.5 mL YT soft agar, 100 microL of X 100 supernatant and 400 microL of overnight culture of JM109 was poured on a YT plate.

Revision as of 06:17, 26 September 2013


Plaque Forming Assay PlacIq

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 PlacIq (constitutive) promoter.

2.Materials and Methods

2-2-0 Plasmid construction

pSB3K3- PlacIq-M13-Plac-GFP (BBa_K1139020)

pSB3K3-M13-Plac-GFP (BBa_K113922)

2-2-1Strain

DH5alpha (E. coli of high competence)

JM109 (F+ strain E. coli)

2-2-2 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

2-2-3Protocol

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 9,000 g 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 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. 2. M13 phage genes and M13 origin worked together with lacIq promoter and pSB origin.

(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 9,000 g. Mixture of 3.5 mL YT soft agar, 100 microL of X 100 supernatant and 400 microL of overnight culture of JM109 was poured on a YT plate.

(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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