Team:Tokyo Tech/Experiment/pSB-M13 Plasmid Assay

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<p style="line-height:0em; text-indent:0em;" name="top">pSB-M13 Plasmid Assay</p>
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<h1>Plaque Forming Assay Plux</h1>
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<h1>1. Introduction</h1>
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<h3>1.Introduction
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</h3>
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<h2>
<h2>
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<p>It was revealed that M13 phage genes and M13 origin worked together with pSB origin.  Next, we confirmed whether the release of M13 phage particle depends on the induction of <i>g2p</i> expression.  We inserted <i>lux</i> promoter (activated by LuxR-3OC6HSL complex) upstream of <i>g2p</i>, as an inducible 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. 3-3-1).  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（[https://2013.igem.org/Team:Tokyo_Tech/Project/M13_supplement#1._M13_plasmid_construction See more about our plasmid construction]).
</p>
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[[Image:Titech2013_M13_PlacIq_Fig.1.PNG|600px|thumb|center|Fig. 3-3-1. Our designed plasmid constrction]]
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<h4>Fig 1. Flow chart of this experiment.
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<h1>2. Materials and Methods</h1>
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</h4>
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<h3>2-1. Plasmid construction</h3>
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<h5>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-<i>luxR</i> ) to the soft agar.  Mix them in soft agar.  Then, decant the soft agar to YT plate.
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</h5>
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<h3>2.Materials and Method
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</h3>
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<h2>
<h2>
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2-1 Plasmid construction
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<p>pSB3K3-PlacI<sup>q</sup>-M13-Plac-<i>GFP</i> ([http://parts.igem.org/Part:BBa_K1139020 BBa_K1139020])
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<p>pSB3K3-Plux-M13-Plac-<i>GFP</i> (BBa_K1139021)
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</p>
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<p>pSB6A1-Ptet-<i>luxR</i>  
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<p>pSB3K3-M13-Plac-<i>GFP</i> ([http://parts.igem.org/Part:BBa_K1139022 BBa_K1139022])
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</p></h2>
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<h3>2-2. Strains</h3>
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<h2>
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<p>DH5alpha (<i>E.  coli</i> of high competence)
</p>
</p>
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2-2 assay protocol
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<p>JM109 (F+ strain <i>E. coli</i>)
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2-2-0 Strains
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</p></h2>
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<p>DH5alpha (<i>E. coli</i> of high competence)
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<h3>2-3. Media</h3>
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<h2>
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<p>JM109 (F+ strain <i>E. coli</i>)
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<div style="margin-left:80px;">
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LB
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2-2-1 Media
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{| class="wikitable" cellpadding="6"
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<p>LB
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|Bacto tryptone||10 g/L
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|-
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<p>Bacto tryptone 10 g/L
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|Yeast Extract||&nbsp;&nbsp;5 g/L
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<p>Yeast Extract   5 g/L
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|NaCl||10 g/L
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<p>NaCl           10 g/L
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YT plate
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<p>YT soft agar
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{| class="wikitable" cellpadding="6"
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</p>
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|Bacto tryptone||&nbsp;&nbsp;8 g/L
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<p>Bacto tryptone 8 g/L
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</p>
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|Yeast Extract||&nbsp;&nbsp;5 g/L
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<p>Yeast Extract   5 g/L
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</p>
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|NaCl||&nbsp;&nbsp;5 g/L
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<p>NaCl:          5 g/L
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|Agarose||15 g/L
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<p>Agarose:        6 g/L
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</p>
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<p>YT plate
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YT soft agar
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</p>
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{| class="wikitable" cellpadding="6"
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<p>Bacto tryptone 8 g/L
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|Bacto tryptone||8 g/L
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</p>
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|-
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<p>Yeast Extract   5 g/L
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|Yeast Extract||5 g/L
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</p>
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<p>NaCl:          5 g/L
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|NaCl||5 g/L
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<p>Agarose:        15 g/L
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|Agarose||6 g/L
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2-2-2 Others
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<p>3OC6HSL dissolved in DMSO (>100 microM)
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</p>
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<p>Autoclaved pieces of filter paper (about 1.5 cm in diameter)
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</p>
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2-2-3 Protocol
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<p>Preparation
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</p>
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1.Transform DH5alpha with pSB3K3-Plux-M13-Plac-<i>GFP</i> and pSB6A1-Ptet-<i>luxR</i>
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2.Grow overnight culture (Amp + Kan) of the transformed DH5alpha and JM109 at 37°C.
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3.Take 30 microL of the overnight culture into 3 mL LB (Amp + Kan).  (→fresh culture)
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4.Incubate the fresh culture at 37°C for 2 hours.
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5.Add 3 microL of 5 microM 3OC6HSL in DMSO (final concentration: 5 nM) to the fresh culture and incubate at 37°C for 4 hours.
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6.Spin the overnight culture of the transformed DH5alpha at 9,000 g for 1 minute.
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7.Pipette the supernatant into a 1.5 mL tube.
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8.Dilute it 100 times with water. (→ phage-particle-solution)
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<p>Plaque formation
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</p>
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9.Transform JM109 with pSB6A1-Ptet-<i>luxR</i>
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10.Grow overnight culture of the transformed JM109 at 37°C.
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11.Melt YT soft agar using a microwave.
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12.Add ampicillin to the YT soft agar.
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13.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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14.Dispense 400 microL of overnight culture of the transformed JM109 to a 1.5 mL tube.
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15.Into the 1.5 mL tube, add 100 microL of the phage-particle-solution.
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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.
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17.Wait for YT soft agar to solidify at room temperature (for about 5 minutes).
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18.Put an autoclaved piece of filter paper on the plate, and drip 20 mL of 3OC6HSL in DMSO (100 microM, 5 microM or DMSO only) on the piece of filter paper.
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</h2>
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<h3>3.Result
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<h3>2-4. Protocol</h3>
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</h3>
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<h2>
<h2>
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<p>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. 
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<ol>
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</p>
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<em align="center">Preparation</em>
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<p>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 <i>g2p</i> 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.
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<li>Transform DH5alpha with pSB3K3-PlacI<sup>q</sup>-M13.
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</p>
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<li>Grow overnight culture of the transformed DH5alpha and JM109 at 37°C.<br>
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Fig 3. The plaques formed with 3OC6HSL induction (plaque plotted)
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<em align="center">Plaque formation</em>
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<p>The plaques were formed using JM109 (with pSB6A1-Ptet-<i>luxR</i>) 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 9,000 g. A mixture of 3.5 mL YT soft agar, 100 microL of X 100 supernatant and 400 microL of overnight culture of JM109 (with pSB6A1-Ptet-<i>luxR</i>) 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.
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<li>Spin the overnight culture of the transformed DH5alpha at 9,000g for 1 min.
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</p>
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<li>Pipette the supernatant into a 1.5 mL tube.
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Fig 4.  The analysis of distribution of the plaques
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<li>Dilute it 100 times with water.  (-> phage-particle-solution)
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<p>We totalize the distances from the center of the piece of filter paper to each plaque, by using ImageJ.
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<li>Melt YT soft agar using a microwave.  
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</p>
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<li>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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Fig 5.  The distribution histogram of the plaques
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<li>Dispense 400 µL of overnight culture of JM109 to a 1.5 mL tube.
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<p>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.
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<li>Into the 1.5 mL tube, add 100 µL of the phage-particle-solution.
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<li>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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</ol></h2>
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<h1>3. Result</h1>
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<h2>
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<p>The result of the plaque forming assay is showed in Fig. 3-3-2, 3-3-3.  M13 phage genes and M13 origin worked together with <i>lacI<sup>q</sup></i> promoter and pSB origin.
</p>
</p>
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[[Image:Titech2013_M13_PlacIq_A.jpg|600px|thumb|center|Fig. 3-3-2. 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 9,000g. A mixture of 3.5 mL YT soft agar, 100 µL of 100X supernatant and 400 µL of overnight culture of JM109 was poured on a YT plate.]]
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[[Image:Titech2013_M13_PlacIq_B.jpg|600px|thumb|center|Fig. 3-3-3. A mixture of only 3.5 mL YT soft agar and 500 µL of overnight culture of JM109 was poured on a YT plate.]]
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Latest revision as of 17:14, 28 October 2013


pSB-M13 Plasmid Assay

Contents

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. 3-3-1). Second, we inserted the amplified sequence to pSB3K3 backbone. Finally, upstream of the RBS-g2p, we inserted PlacIq (constitutive) promoter（See more about our plasmid construction).

Fig. 3-3-1. Our designed plasmid constrction

2. Materials and Methods

2-1. Plasmid construction

pSB3K3-PlacIq-M13-Plac-GFP ([http://parts.igem.org/Part:BBa_K1139020 BBa_K1139020])

pSB3K3-M13-Plac-GFP ([http://parts.igem.org/Part:BBa_K1139022 BBa_K1139022])

2-2. Strains

DH5alpha (E. coli of high competence)

JM109 (F+ strain E. coli)

2-3. Media

LB

Bacto tryptone10 g/L
Yeast Extract  5 g/L
NaCl10 g/L

YT plate

Bacto tryptone  8 g/L
Yeast Extract  5 g/L
NaCl  5 g/L
Agarose15 g/L

YT soft agar

Bacto tryptone8 g/L
Yeast Extract5 g/L
NaCl5 g/L
Agarose6 g/L

2-4. 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 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 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.

3. Result

The result of the plaque forming assay is showed in Fig. 3-3-2, 3-3-3. M13 phage genes and M13 origin worked together with lacIq promoter and pSB origin.

Fig. 3-3-2. 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. A mixture of 3.5 mL YT soft agar, 100 µL of 100X supernatant and 400 µL of overnight culture of JM109 was poured on a YT plate.
Fig. 3-3-3. A mixture of only 3.5 mL YT soft agar and 500 µL of overnight culture of JM109 was poured on a YT plate.


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