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| - | <h1>Plaque Forming Assay PlacIq</h1> | + | <h1>pSB-M13 Plasmid Assay</h1> |
| - | <h3>1.Introduction
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| - | </h3>
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| - | <h2>
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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>
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| - | </h2>
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| - | <h3>2.Materials and Method
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| - | </h3>
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| - | <h2>
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| - | 2-2-0 Plasmid construction
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| - | <p>pSB3K3- PlacI<sup>q</sup>-M13-Plac-<i>GFP</i> (BBa_K1139020)
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| - | </p>
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| - | <p>pSB3K3-M13-Plac-<i>GFP</i> (BBa_K113922)
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| - | </p>
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| - | 2-2-1Strain
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| - | <p>DH5alpha (<i>E. coli</i> of high competence)
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| - | </p>
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| - | <p>JM109 (F+ strain <i>E. coli</i>)
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| - | </p>
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| - | 2-2-2 Media
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| - | <p>LB
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| - | </p>
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| - | <p>Bacto tryptone 10 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: 10 g/L
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| - | </p>
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| - | <p>YT plate
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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
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| - | </p>
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| - | <p>Agarose: 15 g/L
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| - | </p>
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| - | 2-2-3Protocol
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| - | <p>Preparation
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| - | </p>
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| - | 1.Transform DH5alpha with pSB3K3- PlacI<sup>q</sup>-M13.
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| - | 2.Grow overnight culture of the transformed DH5alpha and JM109 at 37°C.
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| - | <p>Plaque formation
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| - | </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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| - | 4.Pipette the supernatant into a 1.5 mL tube.
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| - | 5.Dilute it 100 times with water. (→ phage-particle-solution)
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| - | 6.Melt YT soft agar using a microwave.
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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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| - | 8.Dispense 400 microL of overnight culture of JM109 to a 1.5 mL tube.
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| - | 9.Into the 1.5 mL tube, add 100 microL of the phage-particle-solution.
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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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| - | </h2>
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| | <h3> | | <h3> |
| - | 3.Result
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| | </h3> | | </h3> |
| - | <h2>
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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>
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| - | <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.
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| - | </p>
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| - | <p>(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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| - | </p>
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| - | </h2>
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