Difference between revisions of "Team:Kyoto/Experiments"

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    <div>
 
  
−
<h1>Protocols</h1>
+
<!--
−
<h2>Ligation</h2>
+
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 +
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 +
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 +
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 +
<h2>Materials</h2>
  
−
<p>Ligation was performed according to Ligation high Ver.2’s protocol.</p>
+
<table>
 +
<tr>
 +
<th>Kit</th>
 +
<th>Supplier</th>
 +
</tr>
 +
<tr>
 +
<td>Wizard&reg; SV Gel and PCR</td>
 +
<td>Promega</td>
 +
</tr>
 +
<tr>
 +
<td>Ligation high Ver.2</td>
 +
<td>TOYOBO</td>
 +
</tr>
 +
<tr>
 +
<td>FastGene&trade;Plasmid Mini Kit</td>
 +
<td>NIPPON Genetics Co.,Ltd</td>
 +
</tr>
 +
</table>
 +
<table>
 +
<tr>
 +
<th>Enzyme</th>
 +
<th>Supplier</th>
 +
</tr>
 +
<tr>
 +
<td>EcoRI</td>
 +
<td>TaKaRa, Promega</td>
 +
</tr>
 +
<tr>
 +
<td>PstI</td>
 +
<td>TaKaRa, Promega</td>
 +
</tr>
 +
<tr>
 +
<td>SpeI</td>
 +
<td>TaKaRa, Promega</td>
 +
</tr>
 +
<tr>
 +
<td>XbaI</td>
 +
<td>TaKaRa, Promega</td>
 +
</tr>
 +
<tr>
 +
<td>DraII</td>
 +
<td>TaKaRa</td>
 +
</tr>
 +
</table>
  
−
<p>1. unfreeze competent cells on hands. put them on ice.<br>
+
<table>
−
2. add DNA sample (1~20ul) to competent cells. leave them on ice for 30 minutes<br>
+
<tr>
−
3. keep them in heating block 45 seconds, then on ice for 30 minutes.<br>
+
<th>Marker</th>
−
4. add 40~200ul SOC liquid culture medium, then incubate it under shaking culture for 1hour 37℃.<br>
+
<th>Supplier</th>
−
5. seed it onto LB+antibiotics +/- culture. incubate it for 24 hour at 37℃.</p>
+
</tr>
 +
<tr>
 +
<td>1kb DNA Ladder</td>
 +
<td>TaKaRa</td>
 +
</tr>
 +
</table>
  
−
<h2>PCR</h2>
+
<table>
 +
<tr>
 +
<th>Organism</th>
 +
<th>Supplier</th>
 +
</tr>
 +
<tr>
 +
<td>E.coli DH5α Genotype</td>
 +
<td>TaKaRa</td>
 +
</tr>
 +
<tr>
 +
<td>E.coli JM109 Competent Cells</td>
 +
<td>TaKaRa</td>
 +
</tr>
 +
</table>
  
−
<p>Ligation was performed according to Ligation high Ver.2’s protocol.</p>
+
<table>
 +
<tr>
 +
<th>Antibiotics</th>
 +
<th>Supplier</th>
 +
</tr>
 +
<tr>
 +
<td>Chloramphericol</td>
 +
<td>nacalai tesque</td>
 +
</tr>
 +
<tr>
 +
<td>Ampicillin Sodium Salt</td>
 +
<td>nacalai tesque</td>
 +
</tr>
 +
</table>
  
−
<p>1. unfreeze competent cells on hands. put them on ice.<br>
+
<table>
−
2. add DNA sample (1~20ul) to competent cells. leave them on ice for 30 minutes<br>
+
<tr>Equipment Supplier</tr>
−
3. keep them in heating block 45 seconds, then on ice for 30 minutes.<br>
+
<tr>
−
4. add 40~200ul SOC liquid culture medium, then incubate it under shaking culture for 1hour 37℃.<br>
+
<td>BEMLISE SR601</td>
−
5. seed it onto LB+antibiotics +/- culture. incubate it for 24 hour at 37℃.</p>
+
<td>AsahiKASEI</td>
 +
</tr>
 +
<tr>
 +
<td>BioPhotometer</td>
 +
<td>eppendorf</td>
 +
</tr>
 +
<tr>
 +
<td>LABO SHAKER</td>
 +
<td>BIO CRAFT</td>
 +
</tr>
 +
<tr>
 +
<td>CO2 INCUBATOR</td>
 +
<td>SANYO</td>
 +
</tr>
 +
<tr>
 +
<td>Pipette Controller</td>
 +
<td>Biohit Midi Plus</td>
 +
</tr>
 +
<tr>
 +
<td>MiniCentrifuge Model GMC-060</td>
 +
<td>LMS CO.,LTD.</td>
 +
</tr>
 +
<tr>
 +
<td>HIGH-SPEED REFRIGERATED CENTRIFUGE</td>
 +
<td>TOMY</td>
 +
</tr>
 +
<tr>
 +
<td>HIGH-PRESSURE STEAM STERILIZER</td>
 +
<td>TOMY</td>
 +
</tr>
 +
<tr>
 +
<td>VOTEX-GENE2</td>
 +
<td>Scientific Industryes</td>
 +
</tr>
 +
</table>
  
 +
<table>
 +
<tr>
 +
<th>Backbones</th>
 +
<td>Supplier</th>
 +
</tr>
 +
<tr>
 +
<td>pSB1C3</td>
 +
<td>iGEM registry</td>
 +
</tr>
 +
<tr>
 +
<td>pSB1A2</td>
 +
<td>iGEM registry</td>
 +
</tr>
 +
<tr>
 +
<td>pSB3C5</td>
 +
<td>iGEM registry</td>
 +
</tr>
 +
<tr>
 +
<td>BBa_ J61002</td>
 +
<td>iGEM registry</td>
 +
</tr>
 +
</table>
  
 +
<table>
 +
<tr>
 +
<th>Buffer</th>
 +
<th>Supplier</th>
 +
</tr>
 +
<tr>
 +
<td>Sodium Carbonate</td>
 +
<td>Wako</td>
 +
</tr>
 +
<tr>
 +
<td>Sodium Bicarbonate</td>
 +
<td>Wako</td>
 +
</tr>
 +
<tr>
 +
<td>Methanol(99.5%)</td>
 +
<td>Wako</td>
 +
</tr>
 +
<tr>
 +
<td>Sodium Chloride</td>
 +
<td>Wako</td>
 +
</tr>
 +
<tr>
 +
<td>SDS</td>
 +
<td>nacalai tesque</td>
 +
</tr>
 +
<tr>
 +
<td>Trisaminomethane</td>
 +
<td>nacalai tesque</td>
 +
</tr>
 +
<tr>
 +
<td>Potassium dihydrogenphosphste</td>
 +
<td>SIGAMA-ALDRICH</td>
 +
</tr>
 +
<tr>
 +
<td>Potassium Chloride</td>
 +
<td>Wako</td>
 +
</tr>
 +
<tr>
 +
<td>Glycine</td>
 +
<td>Wako</td>
 +
</tr>
 +
<tr>
 +
<td>Agar, Powder</td>
 +
<td>Wako</td>
 +
</tr>
 +
<tr>
 +
<td>Agarose XP</td>
 +
<td>Wako</td>
 +
</tr>
 +
<tr>
 +
<td>10% Hydrochloric Acid</td>
 +
<td>Wako</td>
 +
</tr>
 +
<tr>
 +
<td>Tween-20</td>
 +
<td>SANTA CRUZ</td>
 +
</tr>
 +
</table>
 +
 +
<table>
 +
<tr>
 +
<th>Other Enzyme</th>
 +
<th>Supplier</th>
 +
</tr>
 +
<tr>
 +
<td>KAPA&trade;HiFi HotStart ReadyMix (2x)</td>
 +
<td>KAPABIOSYSTEMS</td>
 +
</tr>
 +
<tr>
 +
<td>KAPA2G&trade; Fast HotStart ReadyMix with dye (2x)</td>
 +
<td>KAPABIOSYSTEMS</td>
 +
</tr>
 +
<tr>
 +
<td>KAPATaq&trade;EXtra HotStart ReadyMix with dye</td>
 +
<td>KAPABIOSYSTEMS</td>
 +
</tr>
 +
<tr>
 +
<td>Quick Taq&reg; HS DyeMix</td>
 +
<td>TOYOBO</td>
 +
</tr>
 +
</table>
 +
 +
<table>
 +
<tr>
 +
<th>SDSPAGE/WB</th>
 +
<th>Supplier</th>
 +
</tr>
 +
<tr>
 +
<td>IMMOBILON - P Blotting Sandwiches</td>
 +
<td>Immobilon</td>
 +
</tr>
 +
<tr>
 +
<td>REAL GEL PLATE (10%)</td>
 +
<td>BIO CRAFT</td>
 +
</tr>
 +
<tr>
 +
<td>BE-210(SDSPAGE)</td>
 +
<td>BIO CRAFT</td>
 +
</tr>
 +
<tr>
 +
<td>BE-330</td>
 +
<td>BIO CRAFT</td>
 +
</tr>
 +
<tr>
 +
<td>Amersham ECL Anti-Mouse IgG</td>
 +
<td>GE healthcare</td>
 +
</tr>
 +
<tr>
 +
<td>Amersham ECL Anti-rabbit IgG</td>
 +
<td>GE healthcare</td>
 +
</tr>
 +
<tr>
 +
<td>G2-4 rabbit anti-serum</td>
 +
<td>Sano</td>
 +
</tr>
 +
<tr>
 +
<td>Amersham ECL Prime Blocking Reagent</td>
 +
<td>GE healthcare</td>
 +
</tr>
 +
<tr>
 +
<td>Amersham ECL Prime WB Detection Reagent</td>
 +
<td>GE healthcare</td>
 +
</tr>
 +
</table>
 +
 +
 +
<h2>Methods</h2>
 +
 +
<h3>Westernblot (Against His tag)</h3>
 +
<ol>
 +
<li>Apply sample to SDS-PAGE minigel (BIOCLAFT 10%).</li>
 +
<li>Soak the gel in transfer buffer</li>
 +
<li>Soak PVDF membrane in 100% methanol for 30 sec</li>
 +
<li>Soak PVDF membrane and filter paper in transfer buffer, leave for 5 min</li>
 +
<li>Set filter paper, membrane, gels, filter paper to semidry transferor in this order from anode to cathode </li>
 +
<li>Transfer the proteins from the gels to the membrane with 100mA for 1 h</li>
 +
<li>Soak membrane in blocking buffer (ECL Blocking reagent 5 g TBST 100ml) for 1 h</li>
 +
<li>Wash for 5 min 3 times with TBST</li>
 +
<li>Apply 1' antibody (anti-His tag or anti-NoVLP 10μl TBST 10 ml) and shake for 1 h</li>
 +
<li>Wash for 5 min 3 times with TBST</li>
 +
<li>Apply 2' antibody (anti-rabbit HRP 4μl:TBST 10ml) and shake for 1 h</li>
 +
<li>Wash for 5 min 3 times</li>
 +
<li>Drain excess wash buffer from the washed membrane and place it protein side up on flat surface</li>
 +
<li>Add detection reagent onto the membrane, covering all of the membrane</li>
 +
<li>Incubate for 5 minutes at room temperature</li>
 +
<li>Drain off excess detection reagent by dabbing with Kimwipe</li>
 +
<li>Place the sample in the CCD camera compartment and record the images</li>
 +
</ol>
 +
 +
<h3>Western Blot (against NoVLP)</h3>
 +
*Sample Preparation*
 +
<ol>
 +
<li>Mix milliQ 12.5ul, SDSlysisbuffer 4.5ul, and VLP.</li>
 +
<li>Mix MilliQ 2.5ul, SDSlysisbuffer 2.5ul, and GE dual protein marker 5ul.</li>
 +
<li>Vortex 1. and 2., then centrifuge them with a minicentrifuge.</li>
 +
<li>Heat the solutions at 95 &deg;C for 5 min.</li>
 +
<li>Vortex the solutions, and centrifuge them again.</li>
 +
</ol>
 +
 +
<h3>Cellulose Affinity Assay</h3>
 +
<ol>
 +
<li>Prepare LB +CP medium that contains objective E.coli, incubate overnight at 37&deg;C</li>
 +
<li>Centrifuge this solution at 200g for 10 min. </li>
 +
<li>Collect the pellet, and add Carbonate bicarbonate buffe so that OD600 values come to about 0.2</li>
 +
<li>Take 1ml out of the cell suspension, and measure OD600 values. This OD600 values are &lt;before&gt;.</li>
 +
<li>Incubate the cell suspension with 3cm by 2.5 cm cellulose sheet (BEMLIESE #606) for 1 h</li>
 +
<li>Measure OD600 values of the cell suspension. This OD600 values are &lt;after&gt;.</li>
 +
<li>Transfer the cellulose sheet to a new buffer of the same volume, incubate for 1 hour</li>
 +
<li>Measure OD600 values of the buffer This OD600 values are &lt;wash&gt;.</li>
 +
</ol>
 +
 +
<h3>Growth Curve Drawing</h3>
 +
<ol>
 +
<li>Measure OD600 of LB liquid medium (10μg/ml CP) and set blank.</li>
 +
<li>Prepare 10ml LB liquid medium (10μg/ml CP) with E.coli sample whose OD600 values are adjusted to 1.0, Cover the tube with
 +
a plastic sheet with airholes</li>
 +
<li>Start incubating at 160rpm, 37&deg;C. Measure the absorbance promptly and set it 0 minute.</li>
 +
<li>Measure OD600 every 30 minutes (before 2hours)/every an hour (after 2hours). </li>
 +
</ol>
 +
 +
<h3>RT-PCR</h3>
 +
<p>RT-PCR was performed using QuantiTect&reg; Reverse Transcription Kit according to the manufacturer's protocol. </p>
 +
 +
<h3>Scanning Electron Microscopy (2015 Summer Experiment)</h3>
 +
<table>
 +
<tr>
 +
<th>Samples</th>
 +
<th>Centrifugal Force</th>
 +
</tr>
 +
<tr>
 +
<td>INPNC-His-scFv(pSB3C5) 25ul+VLP2.5ul</td>
 +
<td>200g</td>
 +
</tr>
 +
<tr>
 +
<td>BclA-His-scFv(pSB3C5)25ul+VLP2.5ul</td>
 +
<td>200g</td>
 +
</tr>
 +
</table>
 +
 +
<p>*Sample Preparation (E.coli+VLP)*</p>
 +
<ol>
 +
<li>Prepare 45ml of sample E.coli liquid culture whose OD600 values are around 1.0</li>
 +
<li>Take 25ul, and pour into sample tubes.</li>
 +
<li>Centrifuge VLP with MiniCentrifuge. After tapping it, pour 2.5ul into tubes.</li>
 +
<li>Incubate for 24 hours.</li>
 +
<li>Centrifuge at 200g for 10 minutes.</li>
 +
<li>Suspend the pellets in 1ml PBS.</li>
 +
<li>Centrifuge at 200g for 10 minutes.</li>
 +
<li>Remove half of the supernatant, then suspend it again.</li>
 +
</ol>
 +
 +
<h3>Scanning Electron Microscopy (2016 Summer Experiment)</h3>
 +
<table>
 +
<tr>
 +
<th>Samples</th>
 +
<th>Centrifugal Force</th>
 +
</tr>
 +
<tr>
 +
<td>INPNC-His-scFv(pSB3C5) 25ul+VLP2.5ul</td>
 +
<td>3000g</td>
 +
</tr>
 +
<tr>
 +
<td>BclA-His-scFv(pSB3C5)25ul+VLP2.5ul</td>
 +
<td>3000g</td>
 +
</tr>
 +
<tr>
 +
<td>INPNC-His-ctl 25ul+VLP2.5ul</td>
 +
<td>3000g</td>
 +
</tr>
 +
<tr>
 +
<td>BclA-His-ctl 25ul+VLP2.5ul</td>
 +
<td>3000g</td>
 +
</tr>
 +
<tr>
 +
<td>INPNC-His-scFv(pSB3C5) 25ul+VLP2.5ul</td>
 +
<td>200g</td>
 +
</tr>
 +
<tr>
 +
<td>BclA-His-scFv(pSB3C5)25ul+VLP2.5ul</td>
 +
<td>200g</td>
 +
</tr>
 +
<tr>
 +
<td>INPNC-His-ctl 25ul+VLP2.5ul</td>
 +
<td>200g</td>
 +
</tr>
 +
<tr>
 +
<td>BclA-His-ctl 25ul+VLP2.5ul</td>
 +
<td>200g</td>
 +
</tr>
 +
</table>
 +
<p>*Sample Preparation (E.coli+VLP)*</p>
 +
<ol>
 +
<li>Prepare 45ml of sample E.coli liquid culture whose OD600 values are adjusted to 1.0</li>
 +
<li>Take 25ul, and pour into sample tubes.</li>
 +
<li>Centrifuge VLP with MiniCentrifuge. After tapping it, pour 2.5ul into tubes.</li>
 +
<li>Incubate for 24 hours.</li>
 +
<li>Centrifuge at 200g/3000g for 10 minutes.</li>
 +
<li>Suspend the pellets in 1ml PBS.</li>
 +
<li>Centrifuge at 200g/3000g for 10 minutes.</li>
 +
<li>Remove half of the supernatant, then suspend it again.</li>
 +
</ol>
 +
<p>*Sample Preparation (PBS+VLP)*</p>
 +
<ol>
 +
<li>After tapping VLP, pour 2ul into 200ml PBS.</li>
 +
<li>Mark where pellets should have aggregated if E. coli were in the tube.</li>
 +
<li>Centrifuge at 200g for 10 minutes.</li>
 +
<li>Take 100ul of the supernatant, then pour into 1. tube.</li>
 +
</ol>
 +
 +
<h3>Miniprep</h3>
 +
<p>Miniprep was performed using FastGene&trade;Plasmid Mini Kit according to the manufacturer's protocols.</p>
 +
 +
<h3>Gel Extraction</h3>
 +
<p>Gel Extraction was performed using FastGene&trade;Plasmid Mini Kit(GP3→MilliQ), and　Wizard&reg; SV Gel and PCR according to the manufacturer's
 +
protocols.
 +
</p>
 +
 +
<h3>Restriction Enzyme Digestion</h3>
 +
<p>Restriction enzyme treatment was performed using Takara Bio's or Promega's restriction enzymes according to the respective
 +
manufacturer's protocols.</p>
 +
 +
<h3>Ligation</h3>
 +
<p>Ligation was performed using Takara Bio's Ligation High Ver.2 according to the manufacturer's protocols.</p>
 +
 +
<h3>Transformation</h3>
 +
<ol>
 +
<li>Thaw competent cells on ice.</li>
 +
<li>Add DNA sample (1~20ul) to competent cells. leave them on ice for 30 minutes</li>
 +
<li>Keep them in heating block for 45 seconds, then cool on ice for 2 minutes.</li>
 +
<li>Add 40~200ul SOC liquid culture medium, then incubate under shaking culture for 1hour 37&deg;C.</li>
 +
<li>Seed it onto LB+antibiotics agar culture. Incubate for 24 hours at 37&deg;C.</li>
 +
</ol>
 +
 +
<h3>PCR</h3>
 +
<p>PCR was performed using Wizard&reg; SV Gel and PCR, KAPA&trade;HiFi HotStart ReadyMix (2x) according to the manufacturer's protocols</p>
 +
 +
<h3>Westernblotting (Membrane fraction)</h3>
 +
<p>Sample Preparation</p>
 +
<ol>
 +
<li>Cultivate 100ml of E. coli culture whose OD600 values are 0.5</li>
 +
<li>Divide the 100ml cell culture in to 50ml tube, centrifuge 5000 rpm for 10 min</li>
 +
<li>Wash with PBS 30ml, centrifuge 5000 rpm for 10 min</li>
 +
<li>Resuspend with 50mM Tris(pH8.0) 100mM NaCl 10% glycerol 10ml</li>
 +
<li>Sonicate both tubes</li>
 +
<li>Centrifuge 4000 rpm for 10 min at room temperature</li>
 +
<li>Ultracentrifuge the supernatant at 45000 rpm (around 190000g) at room temperature, then remove the resulting supernatant</li>
 +
<li>Resuspend in guanidine buffer (50mM Tris (8.0) 300mM NaCl 10mM Imidazole 6M Guanidine-HCl 0.1% NP40 1mM DTT) to denature
 +
proteins</li>
 +
<li>Use Ni-NTA beads to purify the target protein</li>
 +
<li>Resuspend the purified protein in SDS-buffer</li>
 +
<li>Follow the protocol for Westernblotting</li>
 +
</ol>
 +
 +
以下：プライマーリスト
 +
 +
 +
<h3>Sequence</h3>
 +
<p>We outsourced the sequencing to Macrogen<br />
 +
<a href="http://www.macrogen-japan.co.jp/cap_seq_0203.php">http://www.macrogen-japan.co.jp/cap_seq_0203.php</a>
 +
</p>
  
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      <h2>header 2</h2>
 
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      <h3>header 3</h3>
 
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      <ul>
 
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        <li>listlistlistlistlist</li>
 
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        <li>listlistlistlistlist</li>
 
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        <li>listlistlistlistlist</li>
 
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        <li>listlistlistlistlist</li>
 
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        <li>listlistlistlistlist</li>
 
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      </ul>
 
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    </div>
 
−
    <div>
 
−
      <h1>header 1</h1>
 
−
      <h2>header 2</h2>
 
−
      <h3>header 3</h3>
 
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Revision as of 14:34, 15 October 2016

Materials

Kit Supplier
Wizard® SV Gel and PCR Promega
Ligation high Ver.2 TOYOBO
FastGene™Plasmid Mini Kit NIPPON Genetics Co.,Ltd
Enzyme Supplier
EcoRI TaKaRa, Promega
PstI TaKaRa, Promega
SpeI TaKaRa, Promega
XbaI TaKaRa, Promega
DraII TaKaRa
Marker Supplier
1kb DNA Ladder TaKaRa
Organism Supplier
E.coli DH5α Genotype TaKaRa
E.coli JM109 Competent Cells TaKaRa
Antibiotics Supplier
Chloramphericol nacalai tesque
Ampicillin Sodium Salt nacalai tesque
Equipment Supplier
BEMLISE SR601 AsahiKASEI
BioPhotometer eppendorf
LABO SHAKER BIO CRAFT
CO2 INCUBATOR SANYO
Pipette Controller Biohit Midi Plus
MiniCentrifuge Model GMC-060 LMS CO.,LTD.
HIGH-SPEED REFRIGERATED CENTRIFUGE TOMY
HIGH-PRESSURE STEAM STERILIZER TOMY
VOTEX-GENE2 Scientific Industryes
Backbones Supplier
pSB1C3 iGEM registry
pSB1A2 iGEM registry
pSB3C5 iGEM registry
BBa_ J61002 iGEM registry
Buffer Supplier
Sodium Carbonate Wako
Sodium Bicarbonate Wako
Methanol(99.5%) Wako
Sodium Chloride Wako
SDS nacalai tesque
Trisaminomethane nacalai tesque
Potassium dihydrogenphosphste SIGAMA-ALDRICH
Potassium Chloride Wako
Glycine Wako
Agar, Powder Wako
Agarose XP Wako
10% Hydrochloric Acid Wako
Tween-20 SANTA CRUZ
Other Enzyme Supplier
KAPA™HiFi HotStart ReadyMix (2x) KAPABIOSYSTEMS
KAPA2G™ Fast HotStart ReadyMix with dye (2x) KAPABIOSYSTEMS
KAPATaq™EXtra HotStart ReadyMix with dye KAPABIOSYSTEMS
Quick Taq® HS DyeMix TOYOBO
SDSPAGE/WB Supplier
IMMOBILON - P Blotting Sandwiches Immobilon
REAL GEL PLATE (10%) BIO CRAFT
BE-210(SDSPAGE) BIO CRAFT
BE-330 BIO CRAFT
Amersham ECL Anti-Mouse IgG GE healthcare
Amersham ECL Anti-rabbit IgG GE healthcare
G2-4 rabbit anti-serum Sano
Amersham ECL Prime Blocking Reagent GE healthcare
Amersham ECL Prime WB Detection Reagent GE healthcare

Methods

Westernblot (Against His tag)

  1. Apply sample to SDS-PAGE minigel (BIOCLAFT 10%).
  2. Soak the gel in transfer buffer
  3. Soak PVDF membrane in 100% methanol for 30 sec
  4. Soak PVDF membrane and filter paper in transfer buffer, leave for 5 min
  5. Set filter paper, membrane, gels, filter paper to semidry transferor in this order from anode to cathode
  6. Transfer the proteins from the gels to the membrane with 100mA for 1 h
  7. Soak membrane in blocking buffer (ECL Blocking reagent 5 g TBST 100ml) for 1 h
  8. Wash for 5 min 3 times with TBST
  9. Apply 1' antibody (anti-His tag or anti-NoVLP 10μl TBST 10 ml) and shake for 1 h
  10. Wash for 5 min 3 times with TBST
  11. Apply 2' antibody (anti-rabbit HRP 4μl:TBST 10ml) and shake for 1 h
  12. Wash for 5 min 3 times
  13. Drain excess wash buffer from the washed membrane and place it protein side up on flat surface
  14. Add detection reagent onto the membrane, covering all of the membrane
  15. Incubate for 5 minutes at room temperature
  16. Drain off excess detection reagent by dabbing with Kimwipe
  17. Place the sample in the CCD camera compartment and record the images

Western Blot (against NoVLP)

*Sample Preparation*
  1. Mix milliQ 12.5ul, SDSlysisbuffer 4.5ul, and VLP.
  2. Mix MilliQ 2.5ul, SDSlysisbuffer 2.5ul, and GE dual protein marker 5ul.
  3. Vortex 1. and 2., then centrifuge them with a minicentrifuge.
  4. Heat the solutions at 95 °C for 5 min.
  5. Vortex the solutions, and centrifuge them again.

Cellulose Affinity Assay

  1. Prepare LB +CP medium that contains objective E.coli, incubate overnight at 37°C
  2. Centrifuge this solution at 200g for 10 min.
  3. Collect the pellet, and add Carbonate bicarbonate buffe so that OD600 values come to about 0.2
  4. Take 1ml out of the cell suspension, and measure OD600 values. This OD600 values are <before>.
  5. Incubate the cell suspension with 3cm by 2.5 cm cellulose sheet (BEMLIESE #606) for 1 h
  6. Measure OD600 values of the cell suspension. This OD600 values are <after>.
  7. Transfer the cellulose sheet to a new buffer of the same volume, incubate for 1 hour
  8. Measure OD600 values of the buffer This OD600 values are <wash>.

Growth Curve Drawing

  1. Measure OD600 of LB liquid medium (10μg/ml CP) and set blank.
  2. Prepare 10ml LB liquid medium (10μg/ml CP) with E.coli sample whose OD600 values are adjusted to 1.0, Cover the tube with a plastic sheet with airholes
  3. Start incubating at 160rpm, 37°C. Measure the absorbance promptly and set it 0 minute.
  4. Measure OD600 every 30 minutes (before 2hours)/every an hour (after 2hours).

RT-PCR

RT-PCR was performed using QuantiTect® Reverse Transcription Kit according to the manufacturer's protocol.

Scanning Electron Microscopy (2015 Summer Experiment)

Samples Centrifugal Force
INPNC-His-scFv(pSB3C5) 25ul+VLP2.5ul 200g
BclA-His-scFv(pSB3C5)25ul+VLP2.5ul 200g

*Sample Preparation (E.coli+VLP)*

  1. Prepare 45ml of sample E.coli liquid culture whose OD600 values are around 1.0
  2. Take 25ul, and pour into sample tubes.
  3. Centrifuge VLP with MiniCentrifuge. After tapping it, pour 2.5ul into tubes.
  4. Incubate for 24 hours.
  5. Centrifuge at 200g for 10 minutes.
  6. Suspend the pellets in 1ml PBS.
  7. Centrifuge at 200g for 10 minutes.
  8. Remove half of the supernatant, then suspend it again.

Scanning Electron Microscopy (2016 Summer Experiment)

Samples Centrifugal Force
INPNC-His-scFv(pSB3C5) 25ul+VLP2.5ul 3000g
BclA-His-scFv(pSB3C5)25ul+VLP2.5ul 3000g
INPNC-His-ctl 25ul+VLP2.5ul 3000g
BclA-His-ctl 25ul+VLP2.5ul 3000g
INPNC-His-scFv(pSB3C5) 25ul+VLP2.5ul 200g
BclA-His-scFv(pSB3C5)25ul+VLP2.5ul 200g
INPNC-His-ctl 25ul+VLP2.5ul 200g
BclA-His-ctl 25ul+VLP2.5ul 200g

*Sample Preparation (E.coli+VLP)*

  1. Prepare 45ml of sample E.coli liquid culture whose OD600 values are adjusted to 1.0
  2. Take 25ul, and pour into sample tubes.
  3. Centrifuge VLP with MiniCentrifuge. After tapping it, pour 2.5ul into tubes.
  4. Incubate for 24 hours.
  5. Centrifuge at 200g/3000g for 10 minutes.
  6. Suspend the pellets in 1ml PBS.
  7. Centrifuge at 200g/3000g for 10 minutes.
  8. Remove half of the supernatant, then suspend it again.

*Sample Preparation (PBS+VLP)*

  1. After tapping VLP, pour 2ul into 200ml PBS.
  2. Mark where pellets should have aggregated if E. coli were in the tube.
  3. Centrifuge at 200g for 10 minutes.
  4. Take 100ul of the supernatant, then pour into 1. tube.

Miniprep

Miniprep was performed using FastGene™Plasmid Mini Kit according to the manufacturer's protocols.

Gel Extraction

Gel Extraction was performed using FastGene™Plasmid Mini Kit(GP3→MilliQ), and　Wizard® SV Gel and PCR according to the manufacturer's protocols.

Restriction Enzyme Digestion

Restriction enzyme treatment was performed using Takara Bio's or Promega's restriction enzymes according to the respective manufacturer's protocols.

Ligation

Ligation was performed using Takara Bio's Ligation High Ver.2 according to the manufacturer's protocols.

Transformation

  1. Thaw competent cells on ice.
  2. Add DNA sample (1~20ul) to competent cells. leave them on ice for 30 minutes
  3. Keep them in heating block for 45 seconds, then cool on ice for 2 minutes.
  4. Add 40~200ul SOC liquid culture medium, then incubate under shaking culture for 1hour 37°C.
  5. Seed it onto LB+antibiotics agar culture. Incubate for 24 hours at 37°C.

PCR

PCR was performed using Wizard® SV Gel and PCR, KAPA™HiFi HotStart ReadyMix (2x) according to the manufacturer's protocols

Westernblotting (Membrane fraction)

Sample Preparation

  1. Cultivate 100ml of E. coli culture whose OD600 values are 0.5
  2. Divide the 100ml cell culture in to 50ml tube, centrifuge 5000 rpm for 10 min
  3. Wash with PBS 30ml, centrifuge 5000 rpm for 10 min
  4. Resuspend with 50mM Tris(pH8.0) 100mM NaCl 10% glycerol 10ml
  5. Sonicate both tubes
  6. Centrifuge 4000 rpm for 10 min at room temperature
  7. Ultracentrifuge the supernatant at 45000 rpm (around 190000g) at room temperature, then remove the resulting supernatant
  8. Resuspend in guanidine buffer (50mM Tris (8.0) 300mM NaCl 10mM Imidazole 6M Guanidine-HCl 0.1% NP40 1mM DTT) to denature proteins
  9. Use Ni-NTA beads to purify the target protein
  10. Resuspend the purified protein in SDS-buffer
  11. Follow the protocol for Westernblotting
以下：プライマーリスト

Sequence

We outsourced the sequencing to Macrogen
http://www.macrogen-japan.co.jp/cap_seq_0203.php