Difference between revisions of "Team:HokkaidoU Japan/Notebook/lcasei"
| Line 12: | Line 12: | ||
<h2 id="august">August</h2> | <h2 id="august">August</h2> | ||
| − | < | + | <h3>2015/08/11</h3> |
<!-- Transformaion（プレ培養なし） --> | <!-- Transformaion（プレ培養なし） --> | ||
| − | < | + | <h4>Transformation</h4> |
| − | <p | + | <p>Mitsumoto, Onoda</p> |
| − | <p | + | <p>pHIL253</p> |
| − | <ol | + | <ol> |
| − | <li>Added | + | <li>Added 5 µL of pHIL253 to µL of thawed competent cells (DH5α) on ice.</li> |
<li>Incubated on ice for 30 min.</li> | <li>Incubated on ice for 30 min.</li> | ||
<li>Heat-shocked for 60 sec at 42℃.</li> | <li>Heat-shocked for 60 sec at 42℃.</li> | ||
<li>Spread 300 µL of the culture onto plate with LBA.</li> | <li>Spread 300 µL of the culture onto plate with LBA.</li> | ||
| − | <li>Incubated the plate at 37℃ for | + | <li>Incubated the plate at 37℃ for 16 hrs.</li> |
</ol> | </ol> | ||
<!-- Transformaion（プレ培養なし） END --> | <!-- Transformaion（プレ培養なし） END --> | ||
| − | < | + | <h3>2015/08/12</h3> |
<!-- Liquid Culture --> | <!-- Liquid Culture --> | ||
| − | < | + | <h4>Liquid Culture</h4> |
| − | <p | + | <p>Mitsumoto</p> |
| − | <p | + | <p>pHIL253</p> |
| − | <table | + | <table> |
<tr><th>Reagent</th><th>Volume</th></tr> | <tr><th>Reagent</th><th>Volume</th></tr> | ||
<tr><td>Single Colony</td><td>-</td></tr> | <tr><td>Single Colony</td><td>-</td></tr> | ||
| Line 39: | Line 39: | ||
<tr><td>Ampicillin</td><td>2 µL</td></tr> | <tr><td>Ampicillin</td><td>2 µL</td></tr> | ||
</table> | </table> | ||
| − | <p | + | <p>Cultured for 20 hrs.</p> |
<!-- Liquid Culture END --> | <!-- Liquid Culture END --> | ||
| − | < | + | <h3>2015/08/13</h3> |
<!-- Mini-prep --> | <!-- Mini-prep --> | ||
| − | < | + | <h4>Mini-prep</h4> |
| − | <p | + | <p>Mitsumoto</p> |
| − | <p | + | <p>pHIL253 |
<br>FastGene<sup>TM</sup> Plasmid mini kit (Nippon Genetics Co., Ltd) | <br>FastGene<sup>TM</sup> Plasmid mini kit (Nippon Genetics Co., Ltd) | ||
| − | <br | + | <br>standard protocol</p> |
<!-- Mini-prep END --> | <!-- Mini-prep END --> | ||
<!-- Preparation of Bacteria --> | <!-- Preparation of Bacteria --> | ||
| − | < | + | <h4>Preparation of Bacteria</h4> |
| − | <p | + | <p>Mitsumoto</p> |
| − | <p | + | <p>AHU1910</p> |
| − | <ol | + | <ol> |
<li>Added 5 mL of <i>L. casei</i> to MRS medium.</li> | <li>Added 5 mL of <i>L. casei</i> to MRS medium.</li> | ||
<li>Cultured overnight.</li> | <li>Cultured overnight.</li> | ||
| Line 72: | Line 72: | ||
<!-- Electroporation --> | <!-- Electroporation --> | ||
| − | < | + | <h4>Electroporation</h4> |
| − | <p | + | <p>Mitsumoto</p> |
| − | <p | + | <p>AHU1910</p> |
| − | <ol | + | <ol> |
<li>Prepared the pHIL253 to 300 ng/10 µL (TE pH 8).</li> | <li>Prepared the pHIL253 to 300 ng/10 µL (TE pH 8).</li> | ||
<li>Mixed 10 µL of plasmid and 200 µL of bacteria and incubated on ice for 10 min.</li> | <li>Mixed 10 µL of plasmid and 200 µL of bacteria and incubated on ice for 10 min.</li> | ||
| Line 85: | Line 85: | ||
| − | < | + | <h3>2015/08/19</h3> |
<!-- Electrophoresis --> | <!-- Electrophoresis --> | ||
| − | < | + | <h4>Electrophoresis</h4> |
| − | <p | + | <p>Mitsumoto</p> |
| − | <p | + | <p>pHIL253</p> |
| − | <table | + | <table> |
<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr> | <tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr> | ||
| − | <tr><td | + | <tr><td>2%</td><td>100V</td><td>30 min</td><td>1/2 x TBE</td></tr> |
</table> | </table> | ||
<!-- Electrophoresis END --> | <!-- Electrophoresis END --> | ||
<!-- PCR 2STEP--> | <!-- PCR 2STEP--> | ||
| − | < | + | <h4>PCR</h4> |
| − | <p | + | <p>Mitsumoto</p> |
| − | <p | + | <p>XbaⅠ - oripAMβ1 - repE - Erythromycin resistant gene - P<sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence - SpeⅠ, proCrp4</p> |
| − | <table | + | <table> |
<tr><th>Reagent</th><th>Volume</th></tr> | <tr><th>Reagent</th><th>Volume</th></tr> | ||
| − | <tr><td | + | <tr><td>pHIL253</td><td>1 µL</td></tr> |
| − | <tr><td | + | <tr><td>XbaⅠ - oripAMβ1 - repE - F - primer 10 µM</td><td>1 µL</td></tr> |
| − | <tr><td | + | <tr><td>SpeⅠ - Sec signal sequence - R - primer 10 µM</td><td>1 µL</td></tr> |
<tr><td>KOD - Plus - Neo</td><td>1 µL</td></tr> | <tr><td>KOD - Plus - Neo</td><td>1 µL</td></tr> | ||
<tr><td>10 × PCR Buffer for KOD - Plus - Neo</td><td>5 µL</td></tr> | <tr><td>10 × PCR Buffer for KOD - Plus - Neo</td><td>5 µL</td></tr> | ||
| Line 113: | Line 113: | ||
<tr><td><b>Total</b></td><td><b>50 µL</b></td></tr> | <tr><td><b>Total</b></td><td><b>50 µL</b></td></tr> | ||
</table> | </table> | ||
| − | <p | + | <p>2 Step Cycle (Tm value ≥ 63℃)</p> |
| − | <table | + | <table> |
<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr> | <tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr> | ||
<tr><td>Start</td><td>94℃</td><td>120 sec</td><td>Initialization</td><td></td></tr> | <tr><td>Start</td><td>94℃</td><td>120 sec</td><td>Initialization</td><td></td></tr> | ||
| − | <tr><td>Cycle 1</td><td>98℃</td><td>10 sec</td><td>Denaturation</td><td | + | <tr><td>Cycle 1</td><td>98℃</td><td>10 sec</td><td>Denaturation</td><td>35 cycle</td></tr> |
| − | <tr><td>Cycle 2</td><td>68℃</td><td | + | <tr><td>Cycle 2</td><td>68℃</td><td>120 sec</td><td>Annealing / Elongation</td><td>35 cycle</td></tr> |
<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr> | <tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr> | ||
</table> | </table> | ||
<!-- PCR 2STEP END --> | <!-- PCR 2STEP END --> | ||
| − | < | + | <h3>2015/08/24</h3> |
<!-- Ligation --> | <!-- Ligation --> | ||
| − | < | + | <h4>Ligation</h4> |
| − | <p | + | <p>Onoda</p> |
| − | <p | + | <p>BBa_E0040 on pSB1A2 / XbaⅠ - oripAMβ1 - repE - Erythromycin resistant gene - P<sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence - SpeⅠ</p> |
| − | <table | + | <table> |
<tr><th>Reagent</th><th>Volume</th></tr> | <tr><th>Reagent</th><th>Volume</th></tr> | ||
| − | <tr><td | + | <tr><td>BBa_E0040 on pSB1A2</td><td>1 µL</td></tr> |
| − | <tr><td | + | <tr><td>XbaⅠ - oripAMβ1 - repE - Erythromycin resistant gene - P<sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence - SpeⅠ</td><td>2 µL</td></tr> |
| − | <tr><td>Mighty Mix</td><td | + | <tr><td>Mighty Mix</td><td>3 µL</td></tr> |
| − | <tr><td>DW</td><td | + | <tr><td>DW</td><td>4 µL</td></tr> |
| − | <tr><td><b>Total</b></td><td><b | + | <tr><td><b>Total</b></td><td><b>10 µL</b></td></tr> |
</table> | </table> | ||
| − | <p | + | <p>Ligation</p> |
| − | <table | + | <table> |
<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr> | <tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr> | ||
<tr><td>1</td><td>16℃</td><td>30 min</td><td>Ligation</td></tr> | <tr><td>1</td><td>16℃</td><td>30 min</td><td>Ligation</td></tr> | ||
| Line 146: | Line 146: | ||
<!-- Ligation --> | <!-- Ligation --> | ||
| − | < | + | <h4>Ligation</h4> |
| − | <p | + | <p>Onoda</p> |
| − | <p | + | <p>BBa_R0040 on pSB1C3 / XbaⅠ - oripAMβ1 - repE - Erythromycin resistant gene - P<sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence - SpeⅠ</p> |
| − | <table | + | <table> |
<tr><th>Reagent</th><th>Volume</th></tr> | <tr><th>Reagent</th><th>Volume</th></tr> | ||
| − | <tr><td | + | <tr><td>BBa_R0040 on pSB1C3</td><td>1 µL</td></tr> |
| − | <tr><td | + | <tr><td>XbaⅠ - oripAMβ1 - repE - Erythromycin resistant gene - P<sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence - SpeⅠ</td><td>2 µL</td></tr> |
| − | <tr><td>Mighty Mix</td><td | + | <tr><td>Mighty Mix</td><td>3 µL</td></tr> |
| − | <tr><td>DW</td><td | + | <tr><td>DW</td><td>4 µL</td></tr> |
| − | <tr><td><b>Total</b></td><td><b | + | <tr><td><b>Total</b></td><td><b>10 µL</b></td></tr> |
</table> | </table> | ||
| − | <p | + | <p>Ligation</p> |
| − | <table | + | <table> |
<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr> | <tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr> | ||
<tr><td>1</td><td>16℃</td><td>30 min</td><td>Ligation</td></tr> | <tr><td>1</td><td>16℃</td><td>30 min</td><td>Ligation</td></tr> | ||
| Line 168: | Line 168: | ||
<!-- Ligation --> | <!-- Ligation --> | ||
| − | < | + | <h4>Ligation</h4> |
| − | <p | + | <p>Onoda</p> |
| − | <p | + | <p>pSB1C3 XbaⅠ & SpeⅠ (Digestion product) / XbaⅠ - oripAMβ1 - repE - Erythromycin resistant gene - P<sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence - SpeⅠ</p> |
| − | <table | + | <table> |
<tr><th>Reagent</th><th>Volume</th></tr> | <tr><th>Reagent</th><th>Volume</th></tr> | ||
| − | <tr><td | + | <tr><td>pSB1C3 XbaⅠ & SpeⅠ (Digestion product)</td><td>1 µL</td></tr> |
| − | <tr><td | + | <tr><td>XbaⅠ - oripAMβ1 - repE - Erythromycin resistant gene - P<sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence - SpeⅠ</td><td>2 µL</td></tr> |
| − | <tr><td>Mighty Mix</td><td | + | <tr><td>Mighty Mix</td><td>3 µL</td></tr> |
| − | <tr><td>DW</td><td | + | <tr><td>DW</td><td>4 µL</td></tr> |
| − | <tr><td><b>Total</b></td><td><b | + | <tr><td><b>Total</b></td><td><b>10 µL</b></td></tr> |
</table> | </table> | ||
| − | <p | + | <p>Ligation</p> |
| − | <table | + | <table> |
<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr> | <tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr> | ||
<tr><td>1</td><td>16℃</td><td>30 min</td><td>Ligation</td></tr> | <tr><td>1</td><td>16℃</td><td>30 min</td><td>Ligation</td></tr> | ||
| Line 189: | Line 189: | ||
<!-- Transformaion（プレ培養あり） --> | <!-- Transformaion（プレ培養あり） --> | ||
| − | < | + | <h4>Transformation</h4> |
| − | <p | + | <p>Onoda</p> |
| − | <p | + | <p>pHIL253, oripAMβ1 - repE - Erythromycin resistant gene - P<sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence - BBa_R0040 on pSB1C3, oripAMβ1 - repE - Erythromycin resistant gene - P<sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence - BBa_E0040 on pSB1A2</p> |
| − | <ol | + | <ol> |
| − | <li>Added | + | <li>Added 1 µL of pHIL253, oripAMβ1 - repE - Erythromycin resistant gene - P<sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence - BBa_R0040 on pSB1C3, oripAMβ1 - repE - Erythromycin resistant gene - P<sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence - BBa_E0040 on pSB1A2 to 50 μL of thawed competent cells (DH5α) on ice.</li> |
<li>Incubated on ice for 30 min.</li> | <li>Incubated on ice for 30 min.</li> | ||
<li>Heat-shocked for 30 sec at 42℃.</li> | <li>Heat-shocked for 30 sec at 42℃.</li> | ||
| − | <li>Added | + | <li>Added 200 μL of LB.</li> |
<li>Incubated the cells for 2 hrs at 37℃.</li> | <li>Incubated the cells for 2 hrs at 37℃.</li> | ||
| − | <li>Spread 300 µL of the culture onto plate with | + | <li>Spread 300 µL of the culture onto plate with LBC.</li> |
| − | <li>Incubated the plate at 37℃ for | + | <li>Incubated the plate at 37℃ for 18 hrs.</li> |
</ol> | </ol> | ||
<!-- Transformaion（プレ培養あり） END --> | <!-- Transformaion（プレ培養あり） END --> | ||
| − | < | + | <h3>2015/08/26</h3> |
<!-- Preparation of Bacteria --> | <!-- Preparation of Bacteria --> | ||
| − | < | + | <h4>Preparation of Bacteria</h4> |
| − | <p | + | <p>Mimata, Sakai</p> |
| − | <p | + | <p>AHU1910</p> |
| − | <ol | + | <ol> |
<li>Added 5 mL of <i>L. casei</i> to MRS medium.</li> | <li>Added 5 mL of <i>L. casei</i> to MRS medium.</li> | ||
<li>Cultured overnight.</li> | <li>Cultured overnight.</li> | ||
| Line 226: | Line 226: | ||
| − | < | + | <h3>2015/08/27</h3> |
<!-- Preparation of Bacteria --> | <!-- Preparation of Bacteria --> | ||
| − | < | + | <h4>Preparation of Bacteria</h4> |
| − | <p | + | <p>Mimata, Sakai</p> |
| − | <p | + | <p>AHU1910</p> |
| − | <ol | + | <ol> |
<li>Added 5 mL of <i>L. casei</i> to MRS medium.</li> | <li>Added 5 mL of <i>L. casei</i> to MRS medium.</li> | ||
<li>Cultured overnight.</li> | <li>Cultured overnight.</li> | ||
| Line 248: | Line 248: | ||
<!-- Electroporation --> | <!-- Electroporation --> | ||
| − | < | + | <h4>Electroporation</h4> |
| − | <p | + | <p>Mimata, Sakai</p> |
| − | <p | + | <p>AHU1910</p> |
| − | <ol | + | <ol> |
<li>Prepared the plasmid to 300 ng/10 µL (TE pH 8).</li> | <li>Prepared the plasmid to 300 ng/10 µL (TE pH 8).</li> | ||
<li>Mixed 10 µL of plasmid and 200 µL of bacteria and incubated on ice for 10 min.</li> | <li>Mixed 10 µL of plasmid and 200 µL of bacteria and incubated on ice for 10 min.</li> | ||
| Line 261: | Line 261: | ||
<!-- Colony PCR 3STEP --> | <!-- Colony PCR 3STEP --> | ||
| − | < | + | <h4>Colony PCR</h4> |
| − | <p | + | <p>Mitsumoto</p> |
| − | <p | + | <p>oripAMβ1 - repE - Erythromycin resistant gene - P<sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence on pSB1C3, oripAMβ1 - repE - Erythromycin resistant gene - P<sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence - BBa_R0040 on pSB1C3, oripAMβ1 - repE - Erythromycin resistant gene - P<sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence BBa_E0040</p> |
| − | <table | + | <table> |
<tr><th>Reagent</th><th>Volume</th></tr> | <tr><th>Reagent</th><th>Volume</th></tr> | ||
<tr><td>Single Colony</td><td>-</td></tr> | <tr><td>Single Colony</td><td>-</td></tr> | ||
| − | <tr><td | + | <tr><td>100UP - EX - F 10 µM</td><td>0.4 µL</td></tr> |
| − | <tr><td | + | <tr><td>200DN - PS - R 10 µM</td><td>0.4 µL</td></tr> |
| − | <tr><td>KAPA Taq</td><td | + | <tr><td>KAPA Taq</td><td>5.0 µL</td></tr> |
| − | <tr><td>DW</td><td | + | <tr><td>DW</td><td>4.2 µL</td></tr> |
| − | <tr><td><b>Total</b></td><td><b | + | <tr><td><b>Total</b></td><td><b>10 µL</b></td></tr> |
</table> | </table> | ||
| − | <p | + | <p>3 Step Cycle (Tm value ≤ 63℃)</p> |
| − | <table | + | <table> |
<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr> | <tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr> | ||
<tr><td>Start</td><td>95℃</td><td>120 sec</td><td>Initialization</td><td></td></tr> | <tr><td>Start</td><td>95℃</td><td>120 sec</td><td>Initialization</td><td></td></tr> | ||
| − | <tr><td>Cycle 1</td><td>95℃</td><td>10 sec</td><td>Denaturation</td><td | + | <tr><td>Cycle 1</td><td>95℃</td><td>10 sec</td><td>Denaturation</td><td>35 cycle</td></tr> |
| − | <tr><td>Cycle 2</td><td | + | <tr><td>Cycle 2</td><td>57.6℃</td><td>30 sec</td><td>Annealing</td><td>35 cycle</td></tr> |
| − | <tr><td>Cycle 3</td><td>68℃</td><td>120 sec</td><td>Elongation</td><td | + | <tr><td>Cycle 3</td><td>68℃</td><td>120 sec</td><td>Elongation</td><td>35 cycle</td></tr> |
<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr> | <tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr> | ||
</table> | </table> | ||
| Line 285: | Line 285: | ||
<!-- Colony PCR 3STEP --> | <!-- Colony PCR 3STEP --> | ||
| − | < | + | <h4>Colony PCR</h4> |
| − | <p | + | <p>Mitsumoto</p> |
| − | <p | + | <p>oripAMβ1 - repE - Erythromycin resistant gene - P<sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence on pSB1C3, oripAMβ1 - repE - Erythromycin resistant gene - P<sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence - BBa_R0040 on pSB1C3, oripAMβ1 - repE - Erythromycin resistant gene - P<sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence BBa_E0040</p> |
| − | <table | + | <table> |
<tr><th>Reagent</th><th>Volume</th></tr> | <tr><th>Reagent</th><th>Volume</th></tr> | ||
<tr><td>Single Colony</td><td>-</td></tr> | <tr><td>Single Colony</td><td>-</td></tr> | ||
| − | <tr><td | + | <tr><td>100UP - EX - F 10 µM</td><td>0.4 μL</td></tr> |
| − | <tr><td | + | <tr><td>SpeⅠ - Sec signal sequence reverse 2 10 µM</td><td>0.4 µL</td></tr> |
| − | <tr><td>KAPA Taq</td><td | + | <tr><td>KAPA Taq</td><td>5.0 µL</td></tr> |
| − | <tr><td>DW</td><td | + | <tr><td>DW</td><td>4.2 µL</td></tr> |
| − | <tr><td><b>Total</b></td><td><b | + | <tr><td><b>Total</b></td><td><b>10 µL</b></td></tr> |
</table> | </table> | ||
| − | <p | + | <p>3 Step Cycle (Tm value ≤ 63℃)</p> |
| − | <table | + | <table> |
<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr> | <tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr> | ||
<tr><td>Start</td><td>95℃</td><td>120 sec</td><td>Initialization</td><td></td></tr> | <tr><td>Start</td><td>95℃</td><td>120 sec</td><td>Initialization</td><td></td></tr> | ||
| − | <tr><td>Cycle 1</td><td>95℃</td><td>10 sec</td><td>Denaturation</td><td | + | <tr><td>Cycle 1</td><td>95℃</td><td>10 sec</td><td>Denaturation</td><td>35 cycle</td></tr> |
| − | <tr><td>Cycle 2</td><td | + | <tr><td>Cycle 2</td><td>57.6℃</td><td>30 sec</td><td>Annealing</td><td>35 cycle</td></tr> |
| − | <tr><td>Cycle 3</td><td>68℃</td><td>120 sec</td><td>Elongation</td><td | + | <tr><td>Cycle 3</td><td>68℃</td><td>120 sec</td><td>Elongation</td><td>35 cycle</td></tr> |
<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr> | <tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr> | ||
</table> | </table> | ||
<!-- Colony PCR 3STEP END --> | <!-- Colony PCR 3STEP END --> | ||
| − | < | + | <h3>2015/08/28</h3> |
<!-- Electrophoresis --> | <!-- Electrophoresis --> | ||
| − | < | + | <h4>Electrophoresis</h4> |
| − | <p | + | <p>Mitsumoto</p> |
| − | <p | + | <p>colony PCR products of oripAMβ1 - repE - Erythromycin resistant gene - P<sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence on pSB1C3, oripAMβ1 - repE - Erythromycin resistant gene - P<sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence - BBa_R0040 on pSB1C3 (Colony PCR product), oripAMβ1 - repE - Erythromycin resistant gene - P<sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence BBa_E0040 (Colony PCR product)</p> |
| − | <table | + | <table> |
<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr> | <tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr> | ||
| − | <tr><td | + | <tr><td>2%</td><td>100</ |
| − | span>V</td><td | + | span>V</td><td>45 min</td><td>1/2 x TBE</td></tr> |
</table> | </table> | ||
<!-- Electrophoresis END --> | <!-- Electrophoresis END --> | ||
| − | < | + | <h3>2015/08/31</h3> |
<!-- Electrophoresis --> | <!-- Electrophoresis --> | ||
| − | < | + | <h4>Electrophoresis</h4> |
| − | <p | + | <p>Onoda</p> |
| − | <p | + | <p>oripAMβ1 - repE - Erythromycin resistant gene - P<sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence - BBa_0040 on pSB1A2(colony PCR product), oripAMβ1 - repE - Erythromycin resistant gene - P<sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence - BBa_R0040 on pSB1C3, oripAMβ1 - repE - Erythromycin resistant gene - P<sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence on pSB1C3 </p> |
| − | <table | + | <table> |
<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr> | <tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr> | ||
| − | <tr><td | + | <tr><td>1%</td><td>100 V</td><td>40 min</td><td>1/2 x TBE</td></tr> |
</table> | </table> | ||
<!-- Electrophoresis END --> | <!-- Electrophoresis END --> | ||
<!-- Dephosphorylation --> | <!-- Dephosphorylation --> | ||
| − | < | + | <h4>Dephosphorylation</h4> |
| − | <p | + | <p>Onoda</p> |
| − | <p | + | <p>BBa_E0040 on pSB1A2, BBa_R0040 on pSB1C3, pSB1C3 XbaⅠ & SpeⅠ (Digestion product) </p> |
| − | <table | + | <table> |
<tr><th>Reagent</th><th>Volume</th></tr> | <tr><th>Reagent</th><th>Volume</th></tr> | ||
| − | <tr><td | + | <tr><td>vector</td><td>10 µL</td></tr> |
| − | <tr><td>Antarctic Phosphatase</td><td | + | <tr><td>Antarctic Phosphatase</td><td>1 µL</td></tr> |
| − | <tr><td>Antarctic Phosphatase Buffer</td><td | + | <tr><td>Antarctic Phosphatase Buffer</td><td>2 µL</td></tr> |
| − | <tr><td>DW</td><td | + | <tr><td>DW</td><td>7 µL</td></tr> |
| − | <tr><td><b>Total</b></td><td><b | + | <tr><td><b>Total</b></td><td><b>20 µL</b></td></tr> |
</table> | </table> | ||
| − | <p | + | <p>Dephosphorylation</p> |
| − | <table | + | <table> |
<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr> | <tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr> | ||
<tr><td>1</td><td>37℃</td><td>30 min</td><td>Dephosphorylation</td></tr> | <tr><td>1</td><td>37℃</td><td>30 min</td><td>Dephosphorylation</td></tr> | ||
| Line 354: | Line 354: | ||
<!-- Electrophoresis --> | <!-- Electrophoresis --> | ||
| − | < | + | <h4>Electrophoresis</h4> |
| − | <p | + | <p>Onoda</p> |
| − | <p | + | <p>BBa_E0040 on pSB1A2, BBa_R0040 on pSB1C3, pSB1C3 XbaⅠ & SpeⅠ (Digestion product)</p> |
| − | <table | + | <table> |
<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr> | <tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr> | ||
| − | <tr><td | + | <tr><td>1%</td><td>100 V</td><td>40 min</td><td>1/2 x TBE</td></tr> |
</table> | </table> | ||
<!-- Electrophoresis END --> | <!-- Electrophoresis END --> | ||
<!-- Gel Extract --> | <!-- Gel Extract --> | ||
| − | < | + | <h4>Gel Extract</h4> |
| − | <p | + | <p>Onoda</p> |
| − | <p | + | <p>BBa_E0040 on pSB1A2, BBa_R0040 on pSB1C3, pSB1C3 XbaⅠ & SpeⅠ (Digestion product) |
<br>FastGene<sup>TM</sup> Gel Extraction kit (Nippon Genetics Co., Ltd) | <br>FastGene<sup>TM</sup> Gel Extraction kit (Nippon Genetics Co., Ltd) | ||
<br>DNA extraction from gel</p> | <br>DNA extraction from gel</p> | ||
| Line 373: | Line 373: | ||
<!-- Digestion --> | <!-- Digestion --> | ||
| − | < | + | <h4>Digestion</h4> |
| − | <p | + | <p>Onoda</p> |
| − | <p | + | <p>oripAMβ1 - repE - Erythromycin resistant gene - P<sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence</p> |
| − | <table | + | <table> |
<tr><th>Reagent</th><th>Volume</th></tr> | <tr><th>Reagent</th><th>Volume</th></tr> | ||
| − | <tr><td | + | <tr><td>oripAMβ1 - repE - Erythromycin resistant gene - P<sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence</td><td>20 µL</td></tr> |
| − | <tr><td | + | <tr><td>XbaⅠ</td><td>1 µL</td></tr> |
| − | <tr><td | + | <tr><td>SpeⅠ - HF</td><td>1 µL</td></tr> |
| − | <tr><td | + | <tr><td>10 × M buffer</td><td>3 µL</td></tr> |
| − | <tr><td | + | <tr><td>DW</td><td>5 µL</td></tr> |
<tr><td><b>Total</b></td><td><b>30 µL</b></td></tr> | <tr><td><b>Total</b></td><td><b>30 µL</b></td></tr> | ||
</table> | </table> | ||
| − | <p | + | <p>Digestion</p> |
| − | <table | + | <table> |
<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr> | <tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr> | ||
<tr><td>1</td><td>37℃</td><td>120 min</td><td>Digestion</tr> | <tr><td>1</td><td>37℃</td><td>120 min</td><td>Digestion</tr> | ||
| Line 395: | Line 395: | ||
<!-- Electrophoresis --> | <!-- Electrophoresis --> | ||
| − | < | + | <h4>Electrophoresis</h4> |
| − | <p | + | <p>Onoda</p> |
| − | <p | + | <p>oripAMβ1 - repE - Erythromycin resistant gene - P<sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence</p> |
| − | <table | + | <table> |
<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr> | <tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr> | ||
| − | <tr><td | + | <tr><td>1%</td><td>100 V</td><td>40 min</td><td>1/2 x TBE</td></tr> |
</table> | </table> | ||
<!-- Electrophoresis END --> | <!-- Electrophoresis END --> | ||
<!-- Gel Extract --> | <!-- Gel Extract --> | ||
| − | < | + | <h4>Gel Extract</h4> |
| − | <p | + | <p>Onoda</p> |
| − | <p | + | <p>oripAMβ1 - repE - Erythromycin resistant gene - P<sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence |
<br>FastGene<sup>TM</sup> Gel Extraction kit (Nippon Genetics Co., Ltd) | <br>FastGene<sup>TM</sup> Gel Extraction kit (Nippon Genetics Co., Ltd) | ||
<br>DNA extraction from gel</p> | <br>DNA extraction from gel</p> | ||
| Line 413: | Line 413: | ||
<!-- Ligation --> | <!-- Ligation --> | ||
| − | < | + | <h4>Ligation</h4> |
| − | <p | + | <p>Onoda</p> |
| − | <p | + | <p>BBa_E0040 on pSB1A2, BBa_R0040 on pSB1C3 / oripAMβ1 - repE - Erythromycin resistant gene - P<sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence</p> |
| − | <table | + | <table> |
<tr><th>Reagent</th><th>Volume</th></tr> | <tr><th>Reagent</th><th>Volume</th></tr> | ||
| − | <tr><td | + | <tr><td>BBa_E0040 on pSB1A2, BBa_R0040 on pSB1C3</td><td>3.5 µL</td></tr> |
| − | <tr><td | + | <tr><td>oripAMβ1 - repE - Erythromycin resistant gene - P<sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence</td><td>8 µL</td></tr> |
| − | <tr><td>Mighty Mix</td><td | + | <tr><td>Mighty Mix</td><td>11.5 µL</td></tr> |
| − | <tr><td>DW</td><td | + | <tr><td>DW</td><td>7 µL</td></tr> |
| − | <tr><td><b>Total</b></td><td><b | + | <tr><td><b>Total</b></td><td><b>30 µL</b></td></tr> |
</table> | </table> | ||
| − | <p | + | <p>Ligation</p> |
| − | <table | + | <table> |
<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr> | <tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr> | ||
<tr><td>1</td><td>16℃</td><td>30 min</td><td>Ligation</td></tr> | <tr><td>1</td><td>16℃</td><td>30 min</td><td>Ligation</td></tr> | ||
| Line 434: | Line 434: | ||
<!-- Ligation --> | <!-- Ligation --> | ||
| − | < | + | <h4>Ligation</h4> |
| − | <p | + | <p>Onoda</p> |
| − | <p | + | <p>pSB1C3 XbaⅠ & SpeⅠ (Digestion product) / oripAMβ1 - repE - Erythromycin resistant gene - P<sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence</p> |
| − | <table | + | <table> |
<tr><th>Reagent</th><th>Volume</th></tr> | <tr><th>Reagent</th><th>Volume</th></tr> | ||
| − | <tr><td | + | <tr><td> pSB1C3 XbaⅠ & SpeⅠ (Digestion product)</td><td>20 µL</td></tr> |
| − | <tr><td | + | <tr><td>oripAMβ1 - repE - Erythromycin resistant gene - P<sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence</td><td>3 µL</td></tr> |
| − | <tr><td>Mighty Mix</td><td | + | <tr><td>Mighty Mix</td><td>23 µL</td></tr> |
| − | <tr><td>DW</td><td | + | <tr><td>DW</td><td>4 µL</td></tr> |
| − | <tr><td><b>Total</b></td><td><b | + | <tr><td><b>Total</b></td><td><b>50 µL</b></td></tr> |
</table> | </table> | ||
| − | <p | + | <p>Ligation</p> |
| − | <table | + | <table> |
<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr> | <tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr> | ||
<tr><td>1</td><td>16℃</td><td>30 min</td><td>Ligation</td></tr> | <tr><td>1</td><td>16℃</td><td>30 min</td><td>Ligation</td></tr> | ||
| Line 456: | Line 456: | ||
<h2 id="september">September</h2> | <h2 id="september">September</h2> | ||
| − | < | + | <h3>2015/09/01</h3> |
<!-- Preparation of Bacteria --> | <!-- Preparation of Bacteria --> | ||
| − | < | + | <h4>Preparation of Bacteria</h4> |
| − | <p | + | <p>Mimata</p> |
| − | <p | + | <p>AHU1910</p> |
| − | <ol | + | <ol> |
<li>Added 5 mL of <i>L. casei</i> to MRS medium.</li> | <li>Added 5 mL of <i>L. casei</i> to MRS medium.</li> | ||
<li>Cultured overnight.</li> | <li>Cultured overnight.</li> | ||
| Line 479: | Line 479: | ||
<!-- Electroporation --> | <!-- Electroporation --> | ||
| − | < | + | <h4>Electroporation</h4> |
| − | <p | + | <p>Mimata, Sakai</p> |
| − | <p | + | <p>AHU1910</p> |
| − | <ol | + | <ol> |
<li>Prepared the plasmid to 300 ng/10 µL (TE pH 8).</li> | <li>Prepared the plasmid to 300 ng/10 µL (TE pH 8).</li> | ||
<li>Mixed 10 µL of plasmid and 200 µL of bacteria and incubated on ice for 10 min.</li> | <li>Mixed 10 µL of plasmid and 200 µL of bacteria and incubated on ice for 10 min.</li> | ||
| Line 492: | Line 492: | ||
| − | < | + | <h3>2015/09/02</h3> |
<!-- Gel Extract --> | <!-- Gel Extract --> | ||
| − | < | + | <h4>Gel Extract</h4> |
| − | <p | + | <p>Mitsumoto</p> |
| − | <p | + | <p>Erythromycin resistant gene EcoRⅠ & SpeⅠ(Digestion product) |
<br>FastGene<sup>TM</sup> Gel/PCR Extraction kit (Nippon Genetics Co., Ltd) | <br>FastGene<sup>TM</sup> Gel/PCR Extraction kit (Nippon Genetics Co., Ltd) | ||
<br>DNA extraction from gel</p> | <br>DNA extraction from gel</p> | ||
| Line 505: | Line 505: | ||
| − | < | + | <h3>2015/09/03</h3> |
| Line 512: | Line 512: | ||
<!-- Electrophoresis --> | <!-- Electrophoresis --> | ||
| − | < | + | <h4>Electrophoresis</h4> |
| − | <p | + | <p>Mitsumoto</p> |
| − | <p | + | <p>oripAMβ1 - repE - Erythromycin resistant gene - P<sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence</p> |
| − | <table | + | <table> |
<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr> | <tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr> | ||
| − | <tr><td | + | <tr><td>1%</td><td>100 V</td><td>40 min</td><td>1/2 x TBE</td></tr> |
</table> | </table> | ||
<!-- Electrophoresis END --> | <!-- Electrophoresis END --> | ||
<!-- Transformaion（プレ培養あり） --> | <!-- Transformaion（プレ培養あり） --> | ||
| − | < | + | <h4>Transformation</h4> |
| − | <p | + | <p>Mitsumoto</p> |
| − | <p | + | <p>Erythromycin resistant gene - BBa_B0015 on pSB1C3, |
P<sub>casei</sub> - B0034 - XbaⅠ / SpeⅠ scar - Sec signal sequence - Crp4 - B0034 - XbaⅠ / SpeⅠ scar - Sec signal sequence - BBa_E0040 - BBa_B0015 on pSB1C3, | P<sub>casei</sub> - B0034 - XbaⅠ / SpeⅠ scar - Sec signal sequence - Crp4 - B0034 - XbaⅠ / SpeⅠ scar - Sec signal sequence - BBa_E0040 - BBa_B0015 on pSB1C3, | ||
| − | P<sub>casei</sub> - B0034 - XbaⅠ / SpeⅠ scar - Sec signal sequence - HD-5 - B0034 - XbaⅠ / SpeⅠ scar - Sec signal sequence - BBa_E0040 - BBa_B0015 on pSB1C3, | + | P<sub>casei</sub> - B0034 - XbaⅠ / SpeⅠ scar - Sec signal sequence - HD-5 - B0034 - XbaⅠ / SpeⅠ scar - Sec signal sequence - BBa_E0040 - BBa_B0015 on pSB1C3, </p> |
| − | <ol | + | <ol> |
| − | <li>Added | + | <li>Added 5 µL of |
Erythromycin resistant gene - BBa_B0015 on pSB1C3, | Erythromycin resistant gene - BBa_B0015 on pSB1C3, | ||
P<sub>casei</sub> - B0034 - XbaⅠ / SpeⅠ scar - Sec signal sequence - Crp4 - B0034 - XbaⅠ / SpeⅠ scar - Sec signal sequence - BBa_E0040 - BBa_B0015 on pSB1C3, | P<sub>casei</sub> - B0034 - XbaⅠ / SpeⅠ scar - Sec signal sequence - Crp4 - B0034 - XbaⅠ / SpeⅠ scar - Sec signal sequence - BBa_E0040 - BBa_B0015 on pSB1C3, | ||
P<sub>casei</sub> - B0034 - XbaⅠ / SpeⅠ scar - Sec signal sequence - HD-5 - B0034 - XbaⅠ / SpeⅠ scar - Sec signal sequence - BBa_E0040 - BBa_B0015 on pSB1C3, | P<sub>casei</sub> - B0034 - XbaⅠ / SpeⅠ scar - Sec signal sequence - HD-5 - B0034 - XbaⅠ / SpeⅠ scar - Sec signal sequence - BBa_E0040 - BBa_B0015 on pSB1C3, | ||
| − | + | to 50 µL of thawed competent cells (DH5α) on ice.</li> | |
<li>Incubated on ice for 30 min.</li> | <li>Incubated on ice for 30 min.</li> | ||
<li>Heat-shocked for 30 sec at 42℃.</li> | <li>Heat-shocked for 30 sec at 42℃.</li> | ||
| − | <li>Added | + | <li>Added 200 µL of LB.</li> |
<li>Incubated the cells for 2 hrs at 37℃.</li> | <li>Incubated the cells for 2 hrs at 37℃.</li> | ||
| − | <li>Spread 300 µL of the culture onto plate with | + | <li>Spread 300 µL of the culture onto plate with LBCp.</li> |
| − | <li>Incubated the plate at 37℃ for | + | <li>Incubated the plate at 37℃ for 2 hrs.</li> |
</ol> | </ol> | ||
<!-- Transformaion（プレ培養あり） END --> | <!-- Transformaion（プレ培養あり） END --> | ||
| Line 546: | Line 546: | ||
<!-- Electrophoresis --> | <!-- Electrophoresis --> | ||
| − | < | + | <h4>Electrophoresis</h4> |
| − | <p | + | <p>Mitsumoto</p> |
| − | <p | + | <p> |
| − | EcoRⅠ - codon optimized - matured - HD-5 - PstⅠ | + | EcoRⅠ - codon optimized - matured - HD-5 - PstⅠ</p> |
| − | <table | + | <table> |
<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr> | <tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr> | ||
| − | <tr><td | + | <tr><td>1%</td><td>100 V</td><td>40 min</td><td>1/2 x TBE</td></tr> |
</table> | </table> | ||
<!-- Electrophoresis END --> | <!-- Electrophoresis END --> | ||
| Line 558: | Line 558: | ||
<!-- Electrophoresis --> | <!-- Electrophoresis --> | ||
| − | < | + | <h4>Electrophoresis</h4> |
| − | <p | + | <p>Mitsumoto</p> |
| − | <p | + | <p>oripAMβ1 - repE - Erythromycin resistant gene - P<sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence</p> |
| − | <table | + | <table> |
<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr> | <tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr> | ||
| − | <tr><td | + | <tr><td>1%</td><td>100 V</td><td>40 min</td><td>1/2 x TBE</td></tr> |
</table> | </table> | ||
<!-- Electrophoresis END --> | <!-- Electrophoresis END --> | ||
<!-- Electrophoresis --> | <!-- Electrophoresis --> | ||
| − | < | + | <h4>Electrophoresis</h4> |
| − | <p | + | <p>Mitsumoto</p> |
| − | <p | + | <p>EcoRⅠ - oripAMB1 - repE - PstⅠ, |
| − | EcoRⅠ - codon optimized - matured - HD-5 - PstⅠ | + | EcoRⅠ - codon optimized - matured - HD-5 - PstⅠ</p> |
| − | <table | + | <table> |
<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr> | <tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr> | ||
| − | <tr><td | + | <tr><td>1%</td><td>100 V</td><td>40 min</td><td>1/2 x TBE</td></tr> |
</table> | </table> | ||
<!-- Electrophoresis END --> | <!-- Electrophoresis END --> | ||
<!-- PCR 2STEP--> | <!-- PCR 2STEP--> | ||
| − | < | + | <h4>PCR</h4> |
| − | <p | + | <p>Ono</p> |
| − | <p | + | <p>Erythromycin resistant gene, pAMβ1, HD-5, Crp4</p> |
| − | <table | + | <table> |
<tr><th>Reagent</th><th>Volume</th></tr> | <tr><th>Reagent</th><th>Volume</th></tr> | ||
| − | <tr><td | + | <tr><td>Erythromycin resistant gene, oripAMβ1, HD-5, Crp4</td><td>1 µL</td></tr> |
| − | <tr><td | + | <tr><td>EX - F - Universal 10 µM</td><td>1 µL</td></tr> |
| − | <tr><td | + | <tr><td>PS - R 10 µM</td><td>1 µL</td></tr> |
<tr><td>KOD - Plus - Neo</td><td>1 µL</td></tr> | <tr><td>KOD - Plus - Neo</td><td>1 µL</td></tr> | ||
<tr><td>10 x PCR Buffer for KOD - Plus - Neo</td><td>5 µL</td></tr> | <tr><td>10 x PCR Buffer for KOD - Plus - Neo</td><td>5 µL</td></tr> | ||
| Line 594: | Line 594: | ||
<tr><td><b>Total</b></td><td><b>50 µL</b></td></tr> | <tr><td><b>Total</b></td><td><b>50 µL</b></td></tr> | ||
</table> | </table> | ||
| − | <p | + | <p>2 Step Cycle (Tm value ≥ 63℃)</p> |
| − | <table | + | <table> |
<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr> | <tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr> | ||
<tr><td>Start</td><td>94℃</td><td>120 sec</td><td>Initialization</td><td></td></tr> | <tr><td>Start</td><td>94℃</td><td>120 sec</td><td>Initialization</td><td></td></tr> | ||
| − | <tr><td>Cycle 1</td><td>98℃</td><td>10 sec</td><td>Denaturation</td><td | + | <tr><td>Cycle 1</td><td>98℃</td><td>10 sec</td><td>Denaturation</td><td>30 cycle</td></tr> |
| − | <tr><td>Cycle 2</td><td>68℃</td><td | + | <tr><td>Cycle 2</td><td>68℃</td><td>30 sec</td><td>Annealing / Elongation</td><td>30 cycle</td></tr> |
<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr> | <tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr> | ||
</table> | </table> | ||
| Line 605: | Line 605: | ||
<!-- Electrophoresis --> | <!-- Electrophoresis --> | ||
| − | < | + | <h4>Electrophoresis</h4> |
| − | <p | + | <p>Nishimura, Ono, Mimata</p> |
| − | <p | + | <p>Crp4, HD-5</p> |
| − | <table | + | <table> |
<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr> | <tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr> | ||
| − | <tr><td | + | <tr><td>1%</td><td>100 V</td><td>50 min</td><td>1/2 x TBE</td></tr> |
</table> | </table> | ||
<!-- Electrophoresis END --> | <!-- Electrophoresis END --> | ||
<!-- Digestion --> | <!-- Digestion --> | ||
| − | < | + | <h4>Digestion</h4> |
| − | <p | + | <p>Nisimura, Ono, Mimata</p> |
| − | <p | + | <p>Erythromycin resistant gene</p> |
| − | <table | + | <table> |
<tr><th>Reagent</th><th>Volume</th></tr> | <tr><th>Reagent</th><th>Volume</th></tr> | ||
| − | <tr><td | + | <tr><td>Erythromycin resistant gene</td><td>60 µL</td></tr> |
| − | <tr><td | + | <tr><td>SpeⅠ</td><td>2 µL</td></tr> |
<tr><td><b>Total</b></td><td><b>62 µL</b></td></tr> | <tr><td><b>Total</b></td><td><b>62 µL</b></td></tr> | ||
</table> | </table> | ||
| − | <p | + | <p>Digestion</p> |
| − | <table | + | <table> |
<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr> | <tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr> | ||
<tr><td>1</td><td>37℃</td><td>60 min</td><td>Digestion</tr> | <tr><td>1</td><td>37℃</td><td>60 min</td><td>Digestion</tr> | ||
| Line 634: | Line 634: | ||
<!-- Electrophoresis --> | <!-- Electrophoresis --> | ||
| − | < | + | <h4>Electrophoresis</h4> |
| − | <p | + | <p>Nishimura, Ono, Mimata</p> |
| − | <p | + | <p>Erythromycin resistant gene</p> |
| − | <table | + | <table> |
<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr> | <tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr> | ||
| − | <tr><td | + | <tr><td>2%</td><td>100 V</td><td>60 min</td><td>1/2 x TBE</td></tr> |
</table> | </table> | ||
<!-- Electrophoresis END --> | <!-- Electrophoresis END --> | ||
| Line 646: | Line 646: | ||
<!-- PCR Purification --> | <!-- PCR Purification --> | ||
| − | < | + | <h4>PCR Purification</h4> |
| − | <p | + | <p>Nishimura, Ono, Mimata</p> |
| − | <p | + | <p>repE, Erythromycin resistant gene, Crp4, HD-5 |
<br>FastGene<sup>TM</sup> Gel/PCR Extraction kit (Nippon Genetics Co., Ltd) | <br>FastGene<sup>TM</sup> Gel/PCR Extraction kit (Nippon Genetics Co., Ltd) | ||
<br>Purification of PCR products</p> | <br>Purification of PCR products</p> | ||
| Line 654: | Line 654: | ||
| − | < | + | <h3>2015/09/04</h3> |
| Line 669: | Line 669: | ||
<!-- Electrophoresis --> | <!-- Electrophoresis --> | ||
| − | < | + | <h4>Electrophoresis</h4> |
| − | <p | + | <p>Nishimura</p> |
| − | <p | + | <p>oripAMβ1, HD-5, Crp-4</p> |
| − | <table | + | <table> |
<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr> | <tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr> | ||
| − | <tr><td | + | <tr><td>1%</td><td>100 V</td><td>60 min</td><td>1/2 x TBE</td></tr> |
</table> | </table> | ||
<!-- Electrophoresis END --> | <!-- Electrophoresis END --> | ||
<!-- Electrophoresis --> | <!-- Electrophoresis --> | ||
| − | < | + | <h4>Electrophoresis</h4> |
| − | <p | + | <p>Nishimura</p> |
| − | <p | + | <p>Erythromycin resistant gene, HD-5</p> |
| − | <table | + | <table> |
<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr> | <tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr> | ||
| − | <tr><td | + | <tr><td>2%</td><td>100 V</td><td>60 min</td><td>1/2 x TBE</td></tr> |
</table> | </table> | ||
<!-- Electrophoresis END --> | <!-- Electrophoresis END --> | ||
| Line 690: | Line 690: | ||
<!-- Transformaion（プレ培養あり） --> | <!-- Transformaion（プレ培養あり） --> | ||
| − | < | + | <h4>Transformation</h4> |
| − | <p | + | <p>Fujita, Mimata</p> |
| − | <p | + | <p>Erythromycin resistant gene - BBa_B0015, BBa_R0010 - BBa_B0034 - Crp4 - BBa_B0015, BBa_R0010 - BBa_B0034 - HD5 - BBa_B0015</p> |
| − | <ol | + | <ol> |
| − | <li>Added | + | <li>Added 5 µL of Erythromycin resistant gene - BBa_B0015, BBa_R0010 - BBa_B0034 - Crp4 - BBa_B0015, BBa_R0010 - BBa_B0034 - HD5 - BBa_B0015 to 50 µL of thawed competent cells (DH5α) on ice.</li> |
<li>Incubated on ice for 30 min.</li> | <li>Incubated on ice for 30 min.</li> | ||
<li>Heat-shocked for 30 sec at 42℃.</li> | <li>Heat-shocked for 30 sec at 42℃.</li> | ||
| − | <li>Added | + | <li>Added 200 µL of LB.</li> |
<li>Incubated the cells for 2 hrs at 37℃.</li> | <li>Incubated the cells for 2 hrs at 37℃.</li> | ||
| − | <li>Spread 300 µL of the culture onto plate with | + | <li>Spread 300 µL of the culture onto plate with LBC.</li> |
| − | <li>Incubated the plate at 37℃ for | + | <li>Incubated the plate at 37℃ for 18 hrs.</li> |
</ol> | </ol> | ||
<!-- Transformaion（プレ培養あり） END --> | <!-- Transformaion（プレ培養あり） END --> | ||
| Line 707: | Line 707: | ||
<!-- Digestion --> | <!-- Digestion --> | ||
| − | < | + | <h4>Digestion</h4> |
| − | <p | + | <p>Fujita, Mimata</p> |
| − | <p | + | <p>oripAMβ1, BBa_R0010 - BBa_B0034 - HD5-BBa_B0015, BBa_R0010 - BBa_B0034 - Crp4 - BBa_B0015</p> |
| − | <table | + | <table> |
<tr><th>Reagent</th><th>Volume</th></tr> | <tr><th>Reagent</th><th>Volume</th></tr> | ||
| − | <tr><td | + | <tr><td>oripAMβ1, BBa_R0010 - BBa_B0034 - HD5-BBa_B0015, BBa_R0010 - BBa_B0034 - Crp4 - BBa_B0015</td><td>20 µL</td></tr> |
| − | <tr><td | + | <tr><td>EcoRⅠ</td><td>1 µL</td></tr> |
| − | <tr><td | + | <tr><td>PstⅠ</td><td>1 µL</td></tr> |
| − | <tr><td | + | <tr><td>10 x H Buffer</td><td>5 µL</td></tr> |
| − | <tr><td | + | <tr><td>DW</td><td>23 µL</td></tr> |
<tr><td><b>Total</b></td><td><b>50 µL</b></td></tr> | <tr><td><b>Total</b></td><td><b>50 µL</b></td></tr> | ||
</table> | </table> | ||
| − | <p | + | <p>Digestion</p> |
| − | <table | + | <table> |
<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr> | <tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr> | ||
<tr><td>1</td><td>37℃</td><td>120 min</td><td>Digestion</tr> | <tr><td>1</td><td>37℃</td><td>120 min</td><td>Digestion</tr> | ||
| Line 731: | Line 731: | ||
<!-- Digestion --> | <!-- Digestion --> | ||
| − | < | + | <h4>Digestion</h4> |
| − | <p | + | <p>Onoda</p> |
| − | <p | + | <p>oripAMβ1 - repE - Erythromycin resistant gene - P<sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence</p> |
| − | <table | + | <table> |
<tr><th>Reagent</th><th>Volume</th></tr> | <tr><th>Reagent</th><th>Volume</th></tr> | ||
| − | <tr><td | + | <tr><td>oripAMβ1 - repE - Erythromycin resistant gene - P<sub>casei</sub> - RBS<sub>casei - Sec signal sequence</td><td>20 µL</td></tr> |
| − | <tr><td | + | <tr><td>XbaⅠ</td><td>1 µL</td></tr> |
| − | <tr><td | + | <tr><td>SpeⅠ</td><td>1 µL</td></tr> |
| − | <tr><td | + | <tr><td>10 x M Buffer</td><td>3 µL</td></tr> |
| − | <tr><td | + | <tr><td>DW</td><td>5 µL</td></tr> |
<tr><td><b>Total</b></td><td><b>30 µL</b></td></tr> | <tr><td><b>Total</b></td><td><b>30 µL</b></td></tr> | ||
</table> | </table> | ||
| − | <p | + | <p>Digestion</p> |
| − | <table | + | <table> |
<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr> | <tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr> | ||
<tr><td>1</td><td>37℃</td><td>120 min</td><td>Digestion</tr> | <tr><td>1</td><td>37℃</td><td>120 min</td><td>Digestion</tr> | ||
| Line 756: | Line 756: | ||
<!-- Electrophoresis --> | <!-- Electrophoresis --> | ||
| − | < | + | <h4>Electrophoresis</h4> |
| − | <p | + | <p>Onoda</p> |
| − | <p | + | <p>oripAMβ1 - repE - Erythromycin resistant gene - P<sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence (Digestion product) </p> |
| − | <table | + | <table> |
<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr> | <tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr> | ||
| − | <tr><td | + | <tr><td>2%</td><td>50 V</td><td>60 min</td><td>1/2 x TBE</td></tr> |
</table> | </table> | ||
<!-- Electrophoresis END --> | <!-- Electrophoresis END --> | ||
| Line 770: | Line 770: | ||
| − | < | + | <h3>2015/09/05</h3> |
| Line 783: | Line 783: | ||
<!-- Colony PCR 3STEP --> | <!-- Colony PCR 3STEP --> | ||
| − | < | + | <h4>Colony PCR</h4> |
| − | <p | + | <p>Fujita, Mimata</p> |
| − | <p | + | <p>Erythromycin resistant gene - BBa_B0015 on pSB1C3, |
P<sub>casei</sub> - BBa_B0034 - Sec signal sequence - Crp4 - BBa_B0034 - Sec signal sequence - BBa_E0040 - BBa_B0015 on pSB1C3, | P<sub>casei</sub> - BBa_B0034 - Sec signal sequence - Crp4 - BBa_B0034 - Sec signal sequence - BBa_E0040 - BBa_B0015 on pSB1C3, | ||
| − | P<sub>casei</sub> - BBa_B0034 - Sec signal sequence - HD5 - BBa_B0034 - Sec signal sequence - BBa_E0040 - BBa_B0015 | + | P<sub>casei</sub> - BBa_B0034 - Sec signal sequence - HD5 - BBa_B0034 - Sec signal sequence - BBa_E0040 - BBa_B0015 |
| − | <table | + | <table> |
<tr><th>Reagent</th><th>Volume</th></tr> | <tr><th>Reagent</th><th>Volume</th></tr> | ||
<tr><td>Single Colony</td><td>-</td></tr> | <tr><td>Single Colony</td><td>-</td></tr> | ||
| − | <tr><td | + | <tr><td>100UP - EX - F 10 µM</td><td>0.4 µL</td></tr> |
| − | <tr><td | + | <tr><td>200DN - PS - R 10 µM</td><td>0.4 µL</td></tr> |
| − | <tr><td>KAPA Taq</td><td | + | <tr><td>KAPA Taq</td><td>5.0 µL</td></tr> |
| − | <tr><td>DW</td><td | + | <tr><td>DW</td><td>4.2 µL</td></tr> |
| − | <tr><td><b>Total</b></td><td><b | + | <tr><td><b>Total</b></td><td><b>10 µL</b></td></tr> |
</table> | </table> | ||
| − | <p | + | <p>3 Step Cycle (Tm value ≤ 63℃)</p> |
| − | <table | + | <table> |
<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr> | <tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr> | ||
<tr><td>Start</td><td>95℃</td><td>120 sec</td><td>Initialization</td><td></td></tr> | <tr><td>Start</td><td>95℃</td><td>120 sec</td><td>Initialization</td><td></td></tr> | ||
| − | <tr><td>Cycle 1</td><td>95℃</td><td>30 sec</td><td>Denaturation</td><td | + | <tr><td>Cycle 1</td><td>95℃</td><td>30 sec</td><td>Denaturation</td><td>35 cycle</td></tr> |
| − | <tr><td>Cycle 2</td><td | + | <tr><td>Cycle 2</td><td>57.6℃</td><td>30 sec</td><td>Annealing</td><td>35 cycle</td></tr> |
| − | <tr><td>Cycle 3</td><td>72℃</td><td>210 sec</td><td>Elongation</td><td | + | <tr><td>Cycle 3</td><td>72℃</td><td>210 sec</td><td>Elongation</td><td>35 cycle</td></tr> |
<tr><td>Finish</td><td>72℃</td><td>120 sec</td><td>Final Elongation</td><td></td></tr> | <tr><td>Finish</td><td>72℃</td><td>120 sec</td><td>Final Elongation</td><td></td></tr> | ||
<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr> | <tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr> | ||
| Line 810: | Line 810: | ||
<!-- Electrophoresis --> | <!-- Electrophoresis --> | ||
| − | < | + | <h4>Electrophoresis</h4> |
| − | <p | + | <p>Fujita, Mimata</p> |
| − | <p | + | <p><p>Erythromycin resistant gene - BBa_B0015 on pSB1C3, |
P<sub>casei</sub> - BBa_B0034 - Sec signal sequence - Crp4 - BBa_B0034 - Sec signal sequence - BBa_E0040 - BBa_B0015 on pSB1C3, | P<sub>casei</sub> - BBa_B0034 - Sec signal sequence - Crp4 - BBa_B0034 - Sec signal sequence - BBa_E0040 - BBa_B0015 on pSB1C3, | ||
| − | P<sub>casei</sub> - BBa_B0034 - Sec signal sequence - HD5 - BBa_B0034 - Sec signal sequence - BBa_E0040 - BBa_B0015 | + | P<sub>casei</sub> - BBa_B0034 - Sec signal sequence - HD5 - BBa_B0034 - Sec signal sequence - BBa_E0040 - BBa_B0015</p> |
| − | <table | + | <table> |
<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr> | <tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr> | ||
| − | <tr><td | + | <tr><td>1%</td><td><span class="kinyuu"100 V</td><td>30 min</td><td>1/2 x TBE</td></tr> |
</table> | </table> | ||
<!-- Electrophoresis END --> | <!-- Electrophoresis END --> | ||
<!-- Mini-prep --> | <!-- Mini-prep --> | ||
| − | < | + | <h4>Mini-prep</h4> |
| − | <p | + | <p>Mimata</p> |
| − | <p | + | <p>Erythromycin resistant gene - BBa_B0015 on pSB1C3, P<sub>casei</sub> - BBa_B0034 - Sec signal sequence - Crp4 - BBa_B0034 - Sec signal sequence - BBa_E0040 - BBa_B0015 on pSB1C3, |
| − | P<sub>casei</sub> - BBa_B0034 - Sec signal sequence - HD5 - BBa_B0034 - Sec signal sequence - BBa_E0040 - BBa_B0015 | + | P<sub>casei</sub> - BBa_B0034 - Sec signal sequence - HD5 - BBa_B0034 - Sec signal sequence - BBa_E0040 - BBa_B0015 |
<br>FastGene<sup>TM</sup> Plasmid mini kit (Nippon Genetics Co., Ltd) | <br>FastGene<sup>TM</sup> Plasmid mini kit (Nippon Genetics Co., Ltd) | ||
| − | <br | + | <br>standard protocol</p> |
<!-- Mini-prep END --> | <!-- Mini-prep END --> | ||
<!-- Ligation --> | <!-- Ligation --> | ||
| − | < | + | <h4>Ligation</h4> |
| − | <p | + | <p>Mimata, Onoda</p> |
| − | <p | + | <p>pSB1C3 / P<sub>casei</sub> - BBa_B0034 - Sec signal sequence - Crp4 - BBa_B0034 - Sec signal sequence - BBa_E0040 - BBa_B0015</p> |
| − | <table | + | <table> |
<tbody><tr><th>Reagent</th><th>Volume</th></tr> | <tbody><tr><th>Reagent</th><th>Volume</th></tr> | ||
| − | <tr><td | + | <tr><td>pSB1C3</td><td>2.5 µL</td></tr> |
| − | <tr><td | + | <tr><td>P<sub>casei</sub> - BBa_B0034 - Sec signal sequence - Crp4 - BBa_B0034 - Sec signal sequence - BBa_E0040 - BBa_B0015</td><td>30 µL</td></tr> |
| − | <tr><td>Mighty Mix</td><td | + | <tr><td>Mighty Mix</td><td>32.5 µL</td></tr> |
| − | <tr><td><b>Total</b></td><td><b | + | <tr><td><b>Total</b></td><td><b>65 µL</b></td></tr> |
</tbody></table> | </tbody></table> | ||
| − | <p | + | <p>Ligation</p> |
| − | <table | + | <table> |
<tbody><tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr> | <tbody><tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr> | ||
<tr><td>1</td><td>16℃</td><td>30 min</td><td>Ligation</td></tr> | <tr><td>1</td><td>16℃</td><td>30 min</td><td>Ligation</td></tr> | ||
| Line 852: | Line 852: | ||
<!-- Ligation --> | <!-- Ligation --> | ||
| − | < | + | <h4>Ligation</h4> |
| − | <p | + | <p>Mimata, Onoda</p> |
| − | <p | + | <p>pSB1C3 / EcoRⅠ - codon optimized - matured HD-5 - PstⅠ</p> |
| − | <table | + | <table> |
<tbody><tr><th>Reagent</th><th>Volume</th></tr> | <tbody><tr><th>Reagent</th><th>Volume</th></tr> | ||
| − | <tr><td | + | <tr><td>pSB1C3</td><td>2.5 µL</td></tr> |
| − | <tr><td | + | <tr><td>EcoRⅠ - codon optimized - matured HD-5 - PstⅠ</td><td>30 µL</td></tr> |
| − | <tr><td>Mighty Mix</td><td | + | <tr><td>Mighty Mix</td><td>32.5 µL</td></tr> |
| − | <tr><td><b>Total</b></td><td><b | + | <tr><td><b>Total</b></td><td><b>65 µL</b></td></tr> |
</tbody></table> | </tbody></table> | ||
| − | <p | + | <p>Ligation</p> |
| − | <table | + | <table> |
<tbody><tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr> | <tbody><tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr> | ||
<tr><td>1</td><td>16℃</td><td>30 min</td><td>Ligation</td></tr> | <tr><td>1</td><td>16℃</td><td>30 min</td><td>Ligation</td></tr> | ||
| Line 873: | Line 873: | ||
<!-- Electrophoresis --> | <!-- Electrophoresis --> | ||
| − | < | + | <h4>Electrophoresis</h4> |
| − | <p | + | <p>Mimata</p> |
| − | <p | + | <p>pHIL253, HD-5 on pSB1C3, Crp4 on pSB1C3</p> |
| − | <table | + | <table> |
<tbody><tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr> | <tbody><tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr> | ||
| − | <tr><td | + | <tr><td>1%</td><td>100 V</td><td>60 min</td><td>1/2 x TBE</td></tr> |
</tbody></table> | </tbody></table> | ||
<!-- Electrophoresis END --> | <!-- Electrophoresis END --> | ||
<!-- Gel Extract --> | <!-- Gel Extract --> | ||
| − | < | + | <h4>Gel Extract</h4> |
| − | <p | + | <p>Mimata</p> |
| − | <p | + | <p>pHIL253 |
<br>FastGene<sup>TM</sup> Gel/PCR Extraction kit (Nippon Genetics Co., Ltd) | <br>FastGene<sup>TM</sup> Gel/PCR Extraction kit (Nippon Genetics Co., Ltd) | ||
<br>DNA extraction from gel</p> | <br>DNA extraction from gel</p> | ||
| Line 892: | Line 892: | ||
<!-- Ligation --> | <!-- Ligation --> | ||
| − | < | + | <h4>Ligation</h4> |
| − | <p | + | <p>Mimata</p> |
| − | <p | + | <p>pSB1C3 / oriAMβ1 - repE - Erythromycin resistant gene - P<sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence</p> |
| − | <table | + | <table> |
<tbody><tr><th>Reagent</th><th>Volume</th></tr> | <tbody><tr><th>Reagent</th><th>Volume</th></tr> | ||
| − | <tr><td | + | <tr><td>pSB1C3</td><td>10 µL</td></tr> |
| − | <tr><td | + | <tr><td>oriAMβ1 - repE - Erythromycin resistant gene - P<sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence</td><td>20 µL</td></tr> |
| − | <tr><td>Mighty Mix</td><td | + | <tr><td>Mighty Mix</td><td>30 µL</td></tr> |
| − | <tr><td><b>Total</b></td><td><b | + | <tr><td><b>Total</b></td><td><b>60 µL</b></td></tr> |
</tbody></table> | </tbody></table> | ||
| − | <p | + | <p>Ligation</p> |
| − | <table | + | <table> |
<tbody><tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr> | <tbody><tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr> | ||
<tr><td>1</td><td>16℃</td><td>30 min</td><td>Ligation</td></tr> | <tr><td>1</td><td>16℃</td><td>30 min</td><td>Ligation</td></tr> | ||
| Line 912: | Line 912: | ||
<!-- Digestion --> | <!-- Digestion --> | ||
| − | < | + | <h4>Digestion</h4> |
| − | <p | + | <p>Sakai</p> |
| − | <p | + | <p>XbaⅠ - oripAMβ1 - repE - Erythromycin resistant gene - SpeⅠ</p> |
| − | <table | + | <table> |
<tr><th>Reagent</th><th>Volume</th></tr> | <tr><th>Reagent</th><th>Volume</th></tr> | ||
| − | <tr><td | + | <tr><td>XbaⅠ - oripAMβ1 - repE - Erythromycin resistant gene - SpeⅠ</td><td>30 µL</td></tr> |
| − | <tr><td | + | <tr><td>XbaⅠ</td><td>2 µL</td></tr> |
| − | <tr><td | + | <tr><td>SpeⅠ</td><td>2 µL</td></tr> |
| − | <tr><td | + | <tr><td>10 x M Buffer</td><td>4 µL</td></tr> |
| − | <tr><td | + | <tr><td>DW</td><td>2 µL</td></tr> |
<tr><td><b>Total</b></td><td><b>40 µL</b></td></tr> | <tr><td><b>Total</b></td><td><b>40 µL</b></td></tr> | ||
</table> | </table> | ||
| − | <p | + | <p>Digestion</p> |
| − | <table | + | <table> |
<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr> | <tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th></tr> | ||
<tr><td>1</td><td>37℃</td><td>120 min</td><td>Digestion</tr> | <tr><td>1</td><td>37℃</td><td>120 min</td><td>Digestion</tr> | ||
| Line 934: | Line 934: | ||
| − | < | + | <h3>2015/09/06</h3> |
| Line 945: | Line 945: | ||
<!-- Colony PCR 3STEP --> | <!-- Colony PCR 3STEP --> | ||
| − | < | + | <h4>Colony PCR</h4> |
| − | <p | + | <p>Fujita</p> |
| − | <p | + | <p>Erythromycin resistant gene - BBa_B0015 on pSB1C3, |
P<sub>casei</sub> - BBa_B0034 - Sec signal sequence - Crp4 - BBa_B0034 - Sec signal sequence - BBa_E0040 - BBa_B0015 on pSB1C3, | P<sub>casei</sub> - BBa_B0034 - Sec signal sequence - Crp4 - BBa_B0034 - Sec signal sequence - BBa_E0040 - BBa_B0015 on pSB1C3, | ||
| − | P<sub>casei</sub> - BBa_B0034 - Sec signal sequence - HD5 - BBa_B0034 - Sec signal sequence - BBa_E0040 - BBa_B0015 | + | P<sub>casei</sub> - BBa_B0034 - Sec signal sequence - HD5 - BBa_B0034 - Sec signal sequence - BBa_E0040 - BBa_B0015</p> |
| − | <table | + | <table> |
<tr><th>Reagent</th><th>Volume</th></tr> | <tr><th>Reagent</th><th>Volume</th></tr> | ||
<tr><td>Single Colony</td><td>-</td></tr> | <tr><td>Single Colony</td><td>-</td></tr> | ||
| − | <tr><td | + | <tr><td>100UP - EX - F 10 µM</td><td>0.4 µL</td></tr> |
| − | <tr><td | + | <tr><td>200DN - PS - R 10 µM</td><td>0.4 µL</td></tr> |
| − | <tr><td>KAPA Taq</td><td | + | <tr><td>KAPA Taq</td><td>5.0 µL</td></tr> |
| − | <tr><td>DW</td><td | + | <tr><td>DW</td><td>4.2 µL</td></tr> |
| − | <tr><td><b>Total</b></td><td><b | + | <tr><td><b>Total</b></td><td><b>10 µL</b></td></tr> |
</table> | </table> | ||
| − | <p | + | <p>3 Step Cycle (Tm value ≤ 63℃)</p> |
| − | <table | + | <table> |
<tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr> | <tr><th>Step</th><th>Temp.</th><th>Time</th><th>Process</th><th>Cycle</th></tr> | ||
<tr><td>Start</td><td>95℃</td><td>120 sec</td><td>Initialization</td><td></td></tr> | <tr><td>Start</td><td>95℃</td><td>120 sec</td><td>Initialization</td><td></td></tr> | ||
| − | <tr><td>Cycle 1</td><td>95℃</td><td>30 sec</td><td>Denaturation</td><td | + | <tr><td>Cycle 1</td><td>95℃</td><td>30 sec</td><td>Denaturation</td><td>35 cycle</td></tr> |
| − | <tr><td>Cycle 2</td><td | + | <tr><td>Cycle 2</td><td>57.6℃</td><td>30 sec</td><td>Annealing</td><td>35 cycle</td></tr> |
| − | <tr><td>Cycle 3</td><td>72℃</td><td>210 sec</td><td>Elongation</td><td | + | <tr><td>Cycle 3</td><td>72℃</td><td>210 sec</td><td>Elongation</td><td>35 cycle</td></tr> |
<tr><td>Finish</td><td>72℃</td><td>120 sec</td><td>Final Elongation</td><td></td></tr> | <tr><td>Finish</td><td>72℃</td><td>120 sec</td><td>Final Elongation</td><td></td></tr> | ||
<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr> | <tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td><td></td></tr> | ||
| Line 973: | Line 973: | ||
<!-- Electrophoresis --> | <!-- Electrophoresis --> | ||
| − | < | + | <h4>Electrophoresis</h4> |
| − | <p | + | <p>Fujita</p> |
| − | <p | + | <p>Erythromycin resistant gene - BBa_B0015 on pSB1C3, |
P<sub>casei</sub> - BBa_B0034 - Sec signal sequence - Crp4 - BBa_B0034 - Sec signal sequence - BBa_E0040 - BBa_B0015 on pSB1C3, | P<sub>casei</sub> - BBa_B0034 - Sec signal sequence - Crp4 - BBa_B0034 - Sec signal sequence - BBa_E0040 - BBa_B0015 on pSB1C3, | ||
| − | P<sub>casei</sub> - BBa_B0034 - Sec signal sequence - HD5 - BBa_B0034 - Sec signal sequence - BBa_E0040 - BBa_B0015 (Colony PCR product) | + | P<sub>casei</sub> - BBa_B0034 - Sec signal sequence - HD5 - BBa_B0034 - Sec signal sequence - BBa_E0040 - BBa_B0015 (Colony PCR product)</p> |
| − | <table | + | <table> |
<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr> | <tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr> | ||
| − | <tr><td | + | <tr><td>1%</td><td>100 V</td><td>30 min</td><td>1/2 x TBE</td></tr> |
</table> | </table> | ||
<!-- Electrophoresis END --> | <!-- Electrophoresis END --> | ||
| Line 988: | Line 988: | ||
| − | < | + | <h3>2015/09/07</h3> |
| Line 998: | Line 998: | ||
<!-- Transformaion--> | <!-- Transformaion--> | ||
| − | < | + | <h4>Transformation</h4> |
| − | <p | + | <p>Sakai</p> |
| − | <p | + | <p>oripAMβ1 - repE - Erythromycin resistant gene - P<sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence on pSB1C3</p> |
| − | <ol | + | <ol> |
| − | <li>Added | + | <li>Added 1.0 µL of oripAMβ1 - repE - Erythromycin resistant gene - P<sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence on pSB1C3 to 50 µL of thawed competent cells (JM1O9) on ice.</li> |
<li>Incubated on ice for 30 min.</li> | <li>Incubated on ice for 30 min.</li> | ||
<li>Heat-shocked for 60 sec at 42℃.</li> | <li>Heat-shocked for 60 sec at 42℃.</li> | ||
| − | <li>Added | + | <li>Added 200 µL of LB.</li> |
<li>Incubated the cells for 2 hrs at 37℃.</li> | <li>Incubated the cells for 2 hrs at 37℃.</li> | ||
| − | <li>Spread 300 µL of the culture onto plate with | + | <li>Spread 300 µL of the culture onto plate with LBC.</li> |
| − | <li>Incubated the plate at 37℃ for | + | <li>Incubated the plate at 37℃ for 12 hrs.</li> |
</ol> | </ol> | ||
<!-- Transformaion END --> | <!-- Transformaion END --> | ||
Latest revision as of 03:53, 19 September 2015
L. casei
August
2015/08/11
Transformation
Mitsumoto, Onoda
pHIL253
- Added 5 µL of pHIL253 to µL of thawed competent cells (DH5α) on ice.
- Incubated on ice for 30 min.
- Heat-shocked for 60 sec at 42℃.
- Spread 300 µL of the culture onto plate with LBA.
- Incubated the plate at 37℃ for 16 hrs.
2015/08/12
Liquid Culture
Mitsumoto
pHIL253
| Reagent | Volume |
|---|---|
| Single Colony | - |
| LB | 2000 µL |
| Ampicillin | 2 µL |
Cultured for 20 hrs.
2015/08/13
Mini-prep
Mitsumoto
pHIL253
FastGeneTM Plasmid mini kit (Nippon Genetics Co., Ltd)
standard protocol
Preparation of Bacteria
Mitsumoto
AHU1910
- Added 5 mL of L. casei to MRS medium.
- Cultured overnight.
- Measured OD600 and diluted with MRS medium to set OD600 to 0.2.
- Stand cultured for 1.5 ~ 2 hrs until OD600 is 0.4 ~ 0.5.
- Incubated the cells on ice for 10 min.
- Centrifuged at 6,000 g for 15 min at 4℃.
- Removed supernatant and added 30 mL of cooled PEB.
- Centrifuged at 6,000 g for 15 min at 4℃.
- Removed supernatant and added 30 mL of cooled PEB.
- Centrifuged at 6,000 g for 15 min at 4℃.
- Removed supernatant and added 1 mL of cooled PEB.
Electroporation
Mitsumoto
AHU1910
- Prepared the pHIL253 to 300 ng/10 µL (TE pH 8).
- Mixed 10 µL of plasmid and 200 µL of bacteria and incubated on ice for 10 min.
- Electoroporated at 2 kV with Gene Pulser (Bio-Rad) and Gene Pulser®/MicroPulserTM Electroporation Cuvettes (0.2 cm gap #1652086).
- Added 800 µL of cooled MRS medium and stand cultured for 3 hrs in 30℃.
- Spread on MRS plate (Em 5 µg/mL), (Amp 5 µg/mL).
2015/08/19
Electrophoresis
Mitsumoto
pHIL253
| Gel Concentration | Voltage | Time | Buffer |
|---|---|---|---|
| 2% | 100V | 30 min | 1/2 x TBE |
PCR
Mitsumoto
XbaⅠ - oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence - SpeⅠ, proCrp4
| Reagent | Volume |
|---|---|
| pHIL253 | 1 µL |
| XbaⅠ - oripAMβ1 - repE - F - primer 10 µM | 1 µL |
| SpeⅠ - Sec signal sequence - R - primer 10 µM | 1 µL |
| KOD - Plus - Neo | 1 µL |
| 10 × PCR Buffer for KOD - Plus - Neo | 5 µL |
| 2 mM dNTPs | 5 µL |
| 25 mM MgSO4 | 3 µL |
| DW | 33 µL |
| Total | 50 µL |
2 Step Cycle (Tm value ≥ 63℃)
| Step | Temp. | Time | Process | Cycle |
|---|---|---|---|---|
| Start | 94℃ | 120 sec | Initialization | |
| Cycle 1 | 98℃ | 10 sec | Denaturation | 35 cycle |
| Cycle 2 | 68℃ | 120 sec | Annealing / Elongation | 35 cycle |
| Store | 4℃ | Hold | Store |
2015/08/24
Ligation
Onoda
BBa_E0040 on pSB1A2 / XbaⅠ - oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence - SpeⅠ
| Reagent | Volume |
|---|---|
| BBa_E0040 on pSB1A2 | 1 µL |
| XbaⅠ - oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence - SpeⅠ | 2 µL |
| Mighty Mix | 3 µL |
| DW | 4 µL |
| Total | 10 µL |
Ligation
| Step | Temp. | Time | Process |
|---|---|---|---|
| 1 | 16℃ | 30 min | Ligation |
| 2 | 65℃ | 10 min | Inactivation |
| Store | 4℃ | Hold | Store |
Ligation
Onoda
BBa_R0040 on pSB1C3 / XbaⅠ - oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence - SpeⅠ
| Reagent | Volume |
|---|---|
| BBa_R0040 on pSB1C3 | 1 µL |
| XbaⅠ - oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence - SpeⅠ | 2 µL |
| Mighty Mix | 3 µL |
| DW | 4 µL |
| Total | 10 µL |
Ligation
| Step | Temp. | Time | Process |
|---|---|---|---|
| 1 | 16℃ | 30 min | Ligation |
| 2 | 65℃ | 10 min | Inactivation |
| Store | 4℃ | Hold | Store |
Ligation
Onoda
pSB1C3 XbaⅠ & SpeⅠ (Digestion product) / XbaⅠ - oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence - SpeⅠ
| Reagent | Volume |
|---|---|
| pSB1C3 XbaⅠ & SpeⅠ (Digestion product) | 1 µL |
| XbaⅠ - oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence - SpeⅠ | 2 µL |
| Mighty Mix | 3 µL |
| DW | 4 µL |
| Total | 10 µL |
Ligation
| Step | Temp. | Time | Process |
|---|---|---|---|
| 1 | 16℃ | 30 min | Ligation |
| 2 | 65℃ | 10 min | Inactivation |
| Store | 4℃ | Hold | Store |
Transformation
Onoda
pHIL253, oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence - BBa_R0040 on pSB1C3, oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence - BBa_E0040 on pSB1A2
- Added 1 µL of pHIL253, oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence - BBa_R0040 on pSB1C3, oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence - BBa_E0040 on pSB1A2 to 50 μL of thawed competent cells (DH5α) on ice.
- Incubated on ice for 30 min.
- Heat-shocked for 30 sec at 42℃.
- Added 200 μL of LB.
- Incubated the cells for 2 hrs at 37℃.
- Spread 300 µL of the culture onto plate with LBC.
- Incubated the plate at 37℃ for 18 hrs.
2015/08/26
Preparation of Bacteria
Mimata, Sakai
AHU1910
- Added 5 mL of L. casei to MRS medium.
- Cultured overnight.
- Measured OD600 and diluted with MRS medium to set OD600 to 0.2.
- Stand cultured for 1.5 ~ 2 hrs until OD600 is 0.4 ~ 0.5.
- Incubated the cells on ice for 10 min.
- Centrifuged at 6,000 g for 15 min at 4℃.
- Removed supernatant and added 30 mL of cooled PEB.
- Centrifuged at 6,000 g for 15 min at 4℃.
- Removed supernatant and added 30 mL of cooled PEB.
- Centrifuged at 6,000 g for 15 min at 4℃.
- Removed supernatant and added 1 mL of cooled PEB.
2015/08/27
Preparation of Bacteria
Mimata, Sakai
AHU1910
- Added 5 mL of L. casei to MRS medium.
- Cultured overnight.
- Measured OD600 and diluted with MRS medium to set OD600 to 0.2.
- Stand cultured for 1.5 ~ 2 hrs until OD600 is 0.4 ~ 0.5.
- Incubated the cells on ice for 10 min.
- Centrifuged at 6,000 g for 15 min at 4℃.
- Removed supernatant and added 30 mL of cooled PEB.
- Centrifuged at 6,000 g for 15 min at 4℃.
- Removed supernatant and added 30 mL of cooled PEB.
- Centrifuged at 6,000 g for 15 min at 4℃.
- Removed supernatant and added 1 mL of cooled PEB.
Electroporation
Mimata, Sakai
AHU1910
- Prepared the plasmid to 300 ng/10 µL (TE pH 8).
- Mixed 10 µL of plasmid and 200 µL of bacteria and incubated on ice for 10 min.
- Electoroporated at 2 kV with Gene Pulser (Bio-Rad) and Gene Pulser®/MicroPulserTM Electroporation Cuvettes (0.2 cm gap #1652086).
- Added 800 µL of cooled MRS medium and stand cultured for 3 hrs in 30℃.
- Spread on MRS plate (Em 5 µg/mL).
Colony PCR
Mitsumoto
oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence on pSB1C3, oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence - BBa_R0040 on pSB1C3, oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence BBa_E0040
| Reagent | Volume |
|---|---|
| Single Colony | - |
| 100UP - EX - F 10 µM | 0.4 µL |
| 200DN - PS - R 10 µM | 0.4 µL |
| KAPA Taq | 5.0 µL |
| DW | 4.2 µL |
| Total | 10 µL |
3 Step Cycle (Tm value ≤ 63℃)
| Step | Temp. | Time | Process | Cycle |
|---|---|---|---|---|
| Start | 95℃ | 120 sec | Initialization | |
| Cycle 1 | 95℃ | 10 sec | Denaturation | 35 cycle |
| Cycle 2 | 57.6℃ | 30 sec | Annealing | 35 cycle |
| Cycle 3 | 68℃ | 120 sec | Elongation | 35 cycle |
| Store | 4℃ | Hold | Store |
Colony PCR
Mitsumoto
oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence on pSB1C3, oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence - BBa_R0040 on pSB1C3, oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence BBa_E0040
| Reagent | Volume |
|---|---|
| Single Colony | - |
| 100UP - EX - F 10 µM | 0.4 μL |
| SpeⅠ - Sec signal sequence reverse 2 10 µM | 0.4 µL |
| KAPA Taq | 5.0 µL |
| DW | 4.2 µL |
| Total | 10 µL |
3 Step Cycle (Tm value ≤ 63℃)
| Step | Temp. | Time | Process | Cycle |
|---|---|---|---|---|
| Start | 95℃ | 120 sec | Initialization | |
| Cycle 1 | 95℃ | 10 sec | Denaturation | 35 cycle |
| Cycle 2 | 57.6℃ | 30 sec | Annealing | 35 cycle |
| Cycle 3 | 68℃ | 120 sec | Elongation | 35 cycle |
| Store | 4℃ | Hold | Store |
2015/08/28
Electrophoresis
Mitsumoto
colony PCR products of oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence on pSB1C3, oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence - BBa_R0040 on pSB1C3 (Colony PCR product), oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence BBa_E0040 (Colony PCR product)
| Gel Concentration | Voltage | Time | Buffer |
|---|---|---|---|
| 2% | 100V | 45 min | 1/2 x TBE |
2015/08/31
Electrophoresis
Onoda
oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence - BBa_0040 on pSB1A2(colony PCR product), oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence - BBa_R0040 on pSB1C3, oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence on pSB1C3
| Gel Concentration | Voltage | Time | Buffer |
|---|---|---|---|
| 1% | 100 V | 40 min | 1/2 x TBE |
Dephosphorylation
Onoda
BBa_E0040 on pSB1A2, BBa_R0040 on pSB1C3, pSB1C3 XbaⅠ & SpeⅠ (Digestion product)
| Reagent | Volume |
|---|---|
| vector | 10 µL |
| Antarctic Phosphatase | 1 µL |
| Antarctic Phosphatase Buffer | 2 µL |
| DW | 7 µL |
| Total | 20 µL |
Dephosphorylation
| Step | Temp. | Time | Process |
|---|---|---|---|
| 1 | 37℃ | 30 min | Dephosphorylation |
| 2 | 65℃ | 10 min | Inactivation |
| Store | 4℃ | Hold | Store |
Electrophoresis
Onoda
BBa_E0040 on pSB1A2, BBa_R0040 on pSB1C3, pSB1C3 XbaⅠ & SpeⅠ (Digestion product)
| Gel Concentration | Voltage | Time | Buffer |
|---|---|---|---|
| 1% | 100 V | 40 min | 1/2 x TBE |
Gel Extract
Onoda
BBa_E0040 on pSB1A2, BBa_R0040 on pSB1C3, pSB1C3 XbaⅠ & SpeⅠ (Digestion product)
FastGeneTM Gel Extraction kit (Nippon Genetics Co., Ltd)
DNA extraction from gel
Digestion
Onoda
oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence
| Reagent | Volume |
|---|---|
| oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence | 20 µL |
| XbaⅠ | 1 µL |
| SpeⅠ - HF | 1 µL |
| 10 × M buffer | 3 µL |
| DW | 5 µL |
| Total | 30 µL |
Digestion
| Step | Temp. | Time | Process |
|---|---|---|---|
| 1 | 37℃ | 120 min | Digestion |
| 2 | 80℃ | 20 min | Inactivation |
| Store | 4℃ | Hold | Store |
Electrophoresis
Onoda
oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence
| Gel Concentration | Voltage | Time | Buffer |
|---|---|---|---|
| 1% | 100 V | 40 min | 1/2 x TBE |
Gel Extract
Onoda
oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence
FastGeneTM Gel Extraction kit (Nippon Genetics Co., Ltd)
DNA extraction from gel
Ligation
Onoda
BBa_E0040 on pSB1A2, BBa_R0040 on pSB1C3 / oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence
| Reagent | Volume |
|---|---|
| BBa_E0040 on pSB1A2, BBa_R0040 on pSB1C3 | 3.5 µL |
| oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence | 8 µL |
| Mighty Mix | 11.5 µL |
| DW | 7 µL |
| Total | 30 µL |
Ligation
| Step | Temp. | Time | Process |
|---|---|---|---|
| 1 | 16℃ | 30 min | Ligation |
| 2 | 65℃ | 10 min | Inactivation |
| Store | 4℃ | Hold | Store |
Ligation
Onoda
pSB1C3 XbaⅠ & SpeⅠ (Digestion product) / oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence
| Reagent | Volume |
|---|---|
| pSB1C3 XbaⅠ & SpeⅠ (Digestion product) | 20 µL |
| oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence | 3 µL |
| Mighty Mix | 23 µL |
| DW | 4 µL |
| Total | 50 µL |
Ligation
| Step | Temp. | Time | Process |
|---|---|---|---|
| 1 | 16℃ | 30 min | Ligation |
| 2 | 65℃ | 10 min | Inactivation |
| Store | 4℃ | Hold | Store |
September
2015/09/01
Preparation of Bacteria
Mimata
AHU1910
- Added 5 mL of L. casei to MRS medium.
- Cultured overnight.
- Measured OD600 and diluted with MRS medium to set OD600 to 0.2.
- Stand cultured for 1.5 ~ 2 hrs until OD600 is 0.4 ~ 0.5.
- Incubated the cells on ice for 10 min.
- Centrifuged at 6,000 g for 15 min at 4℃.
- Removed supernatant and added 30 mL of cooled PEB.
- Centrifuged at 6,000 g for 15 min at 4℃.
- Removed supernatant and added 30 mL of cooled PEB.
- Centrifuged at 6,000 g for 15 min at 4℃.
- Removed supernatant and added 1 mL of cooled PEB.
Electroporation
Mimata, Sakai
AHU1910
- Prepared the plasmid to 300 ng/10 µL (TE pH 8).
- Mixed 10 µL of plasmid and 200 µL of bacteria and incubated on ice for 10 min.
- Electoroporated at 2 kV with Gene Pulser (Bio-Rad) and Gene Pulser®/MicroPulserTM Electroporation Cuvettes(0.2 cm gap #1652086).
- Added 800 µL of cooled MRS medium and stand cultured for 3 hrs in 30℃.
- Spread on MRS plate (Em 5 µg/mL).
2015/09/02
Gel Extract
Mitsumoto
Erythromycin resistant gene EcoRⅠ & SpeⅠ(Digestion product)
FastGeneTM Gel/PCR Extraction kit (Nippon Genetics Co., Ltd)
DNA extraction from gel
2015/09/03
Electrophoresis
Mitsumoto
oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence
| Gel Concentration | Voltage | Time | Buffer |
|---|---|---|---|
| 1% | 100 V | 40 min | 1/2 x TBE |
Transformation
Mitsumoto
Erythromycin resistant gene - BBa_B0015 on pSB1C3, Pcasei - B0034 - XbaⅠ / SpeⅠ scar - Sec signal sequence - Crp4 - B0034 - XbaⅠ / SpeⅠ scar - Sec signal sequence - BBa_E0040 - BBa_B0015 on pSB1C3, Pcasei - B0034 - XbaⅠ / SpeⅠ scar - Sec signal sequence - HD-5 - B0034 - XbaⅠ / SpeⅠ scar - Sec signal sequence - BBa_E0040 - BBa_B0015 on pSB1C3,
- Added 5 µL of Erythromycin resistant gene - BBa_B0015 on pSB1C3, Pcasei - B0034 - XbaⅠ / SpeⅠ scar - Sec signal sequence - Crp4 - B0034 - XbaⅠ / SpeⅠ scar - Sec signal sequence - BBa_E0040 - BBa_B0015 on pSB1C3, Pcasei - B0034 - XbaⅠ / SpeⅠ scar - Sec signal sequence - HD-5 - B0034 - XbaⅠ / SpeⅠ scar - Sec signal sequence - BBa_E0040 - BBa_B0015 on pSB1C3, to 50 µL of thawed competent cells (DH5α) on ice.
- Incubated on ice for 30 min.
- Heat-shocked for 30 sec at 42℃.
- Added 200 µL of LB.
- Incubated the cells for 2 hrs at 37℃.
- Spread 300 µL of the culture onto plate with LBCp.
- Incubated the plate at 37℃ for 2 hrs.
Electrophoresis
Mitsumoto
EcoRⅠ - codon optimized - matured - HD-5 - PstⅠ
| Gel Concentration | Voltage | Time | Buffer |
|---|---|---|---|
| 1% | 100 V | 40 min | 1/2 x TBE |
Electrophoresis
Mitsumoto
oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence
| Gel Concentration | Voltage | Time | Buffer |
|---|---|---|---|
| 1% | 100 V | 40 min | 1/2 x TBE |
Electrophoresis
Mitsumoto
EcoRⅠ - oripAMB1 - repE - PstⅠ, EcoRⅠ - codon optimized - matured - HD-5 - PstⅠ
| Gel Concentration | Voltage | Time | Buffer |
|---|---|---|---|
| 1% | 100 V | 40 min | 1/2 x TBE |
PCR
Ono
Erythromycin resistant gene, pAMβ1, HD-5, Crp4
| Reagent | Volume |
|---|---|
| Erythromycin resistant gene, oripAMβ1, HD-5, Crp4 | 1 µL |
| EX - F - Universal 10 µM | 1 µL |
| PS - R 10 µM | 1 µL |
| KOD - Plus - Neo | 1 µL |
| 10 x PCR Buffer for KOD - Plus - Neo | 5 µL |
| 2 mM dNTPs | 5 µL |
| 25 mM MgSO4 | 3 µL |
| DW | 33 µL |
| Total | 50 µL |
2 Step Cycle (Tm value ≥ 63℃)
| Step | Temp. | Time | Process | Cycle |
|---|---|---|---|---|
| Start | 94℃ | 120 sec | Initialization | |
| Cycle 1 | 98℃ | 10 sec | Denaturation | 30 cycle |
| Cycle 2 | 68℃ | 30 sec | Annealing / Elongation | 30 cycle |
| Store | 4℃ | Hold | Store |
Electrophoresis
Nishimura, Ono, Mimata
Crp4, HD-5
| Gel Concentration | Voltage | Time | Buffer |
|---|---|---|---|
| 1% | 100 V | 50 min | 1/2 x TBE |
Digestion
Nisimura, Ono, Mimata
Erythromycin resistant gene
| Reagent | Volume |
|---|---|
| Erythromycin resistant gene | 60 µL |
| SpeⅠ | 2 µL |
| Total | 62 µL |
Digestion
| Step | Temp. | Time | Process |
|---|---|---|---|
| 1 | 37℃ | 60 min | Digestion |
| Store | 4℃ | Hold | Store |
Electrophoresis
Nishimura, Ono, Mimata
Erythromycin resistant gene
| Gel Concentration | Voltage | Time | Buffer |
|---|---|---|---|
| 2% | 100 V | 60 min | 1/2 x TBE |
PCR Purification
Nishimura, Ono, Mimata
repE, Erythromycin resistant gene, Crp4, HD-5
FastGeneTM Gel/PCR Extraction kit (Nippon Genetics Co., Ltd)
Purification of PCR products
2015/09/04
Electrophoresis
Nishimura
oripAMβ1, HD-5, Crp-4
| Gel Concentration | Voltage | Time | Buffer |
|---|---|---|---|
| 1% | 100 V | 60 min | 1/2 x TBE |
Electrophoresis
Nishimura
Erythromycin resistant gene, HD-5
| Gel Concentration | Voltage | Time | Buffer |
|---|---|---|---|
| 2% | 100 V | 60 min | 1/2 x TBE |
Transformation
Fujita, Mimata
Erythromycin resistant gene - BBa_B0015, BBa_R0010 - BBa_B0034 - Crp4 - BBa_B0015, BBa_R0010 - BBa_B0034 - HD5 - BBa_B0015
- Added 5 µL of Erythromycin resistant gene - BBa_B0015, BBa_R0010 - BBa_B0034 - Crp4 - BBa_B0015, BBa_R0010 - BBa_B0034 - HD5 - BBa_B0015 to 50 µL of thawed competent cells (DH5α) on ice.
- Incubated on ice for 30 min.
- Heat-shocked for 30 sec at 42℃.
- Added 200 µL of LB.
- Incubated the cells for 2 hrs at 37℃.
- Spread 300 µL of the culture onto plate with LBC.
- Incubated the plate at 37℃ for 18 hrs.
Digestion
Fujita, Mimata
oripAMβ1, BBa_R0010 - BBa_B0034 - HD5-BBa_B0015, BBa_R0010 - BBa_B0034 - Crp4 - BBa_B0015
| Reagent | Volume |
|---|---|
| oripAMβ1, BBa_R0010 - BBa_B0034 - HD5-BBa_B0015, BBa_R0010 - BBa_B0034 - Crp4 - BBa_B0015 | 20 µL |
| EcoRⅠ | 1 µL |
| PstⅠ | 1 µL |
| 10 x H Buffer | 5 µL |
| DW | 23 µL |
| Total | 50 µL |
Digestion
| Step | Temp. | Time | Process |
|---|---|---|---|
| 1 | 37℃ | 120 min | Digestion |
| 2 | 80℃ | 20 min | Inactivation |
| Store | 4℃ | Hold | Store |
Digestion
Onoda
oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence
| Reagent | Volume |
|---|---|
| oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence | 20 µL |
| XbaⅠ | 1 µL |
| SpeⅠ | 1 µL |
| 10 x M Buffer | 3 µL |
| DW | 5 µL |
| Total | 30 µL |
Digestion
| Step | Temp. | Time | Process |
|---|---|---|---|
| 1 | 37℃ | 120 min | Digestion |
| 2 | 80℃ | 20 min | Inactivation |
| Store | 4℃ | Hold | Store |
Electrophoresis
Onoda
oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence (Digestion product)
| Gel Concentration | Voltage | Time | Buffer |
|---|---|---|---|
| 2% | 50 V | 60 min | 1/2 x TBE |
2015/09/05
Colony PCR
Fujita, Mimata
Erythromycin resistant gene - BBa_B0015 on pSB1C3, Pcasei - BBa_B0034 - Sec signal sequence - Crp4 - BBa_B0034 - Sec signal sequence - BBa_E0040 - BBa_B0015 on pSB1C3, Pcasei - BBa_B0034 - Sec signal sequence - HD5 - BBa_B0034 - Sec signal sequence - BBa_E0040 - BBa_B0015
| Reagent | Volume |
|---|---|
| Single Colony | - |
| 100UP - EX - F 10 µM | 0.4 µL |
| 200DN - PS - R 10 µM | 0.4 µL |
| KAPA Taq | 5.0 µL |
| DW | 4.2 µL |
| Total | 10 µL |
3 Step Cycle (Tm value ≤ 63℃)
| Step | Temp. | Time | Process | Cycle |
|---|---|---|---|---|
| Start | 95℃ | 120 sec | Initialization | |
| Cycle 1 | 95℃ | 30 sec | Denaturation | 35 cycle |
| Cycle 2 | 57.6℃ | 30 sec | Annealing | 35 cycle |
| Cycle 3 | 72℃ | 210 sec | Elongation | 35 cycle |
| Finish | 72℃ | 120 sec | Final Elongation | |
| Store | 4℃ | Hold | Store |
Electrophoresis
Fujita, Mimata
Erythromycin resistant gene - BBa_B0015 on pSB1C3, Pcasei - BBa_B0034 - Sec signal sequence - Crp4 - BBa_B0034 - Sec signal sequence - BBa_E0040 - BBa_B0015 on pSB1C3, Pcasei - BBa_B0034 - Sec signal sequence - HD5 - BBa_B0034 - Sec signal sequence - BBa_E0040 - BBa_B0015
| Gel Concentration | Voltage | Time | Buffer |
|---|---|---|---|
| 1% | 30 min | 1/2 x TBE |
Mini-prep
Mimata
Erythromycin resistant gene - BBa_B0015 on pSB1C3, Pcasei - BBa_B0034 - Sec signal sequence - Crp4 - BBa_B0034 - Sec signal sequence - BBa_E0040 - BBa_B0015 on pSB1C3,
Pcasei - BBa_B0034 - Sec signal sequence - HD5 - BBa_B0034 - Sec signal sequence - BBa_E0040 - BBa_B0015
FastGeneTM Plasmid mini kit (Nippon Genetics Co., Ltd)
standard protocol
Ligation
Mimata, Onoda
pSB1C3 / Pcasei - BBa_B0034 - Sec signal sequence - Crp4 - BBa_B0034 - Sec signal sequence - BBa_E0040 - BBa_B0015
| Reagent | Volume |
|---|---|
| pSB1C3 | 2.5 µL |
| Pcasei - BBa_B0034 - Sec signal sequence - Crp4 - BBa_B0034 - Sec signal sequence - BBa_E0040 - BBa_B0015 | 30 µL |
| Mighty Mix | 32.5 µL |
| Total | 65 µL |
Ligation
| Step | Temp. | Time | Process |
|---|---|---|---|
| 1 | 16℃ | 30 min | Ligation |
| 2 | 70℃ | 10 min | Inactivation |
| Store | 4℃ | Hold | Store |
Ligation
Mimata, Onoda
pSB1C3 / EcoRⅠ - codon optimized - matured HD-5 - PstⅠ
| Reagent | Volume |
|---|---|
| pSB1C3 | 2.5 µL |
| EcoRⅠ - codon optimized - matured HD-5 - PstⅠ | 30 µL |
| Mighty Mix | 32.5 µL |
| Total | 65 µL |
Ligation
| Step | Temp. | Time | Process |
|---|---|---|---|
| 1 | 16℃ | 30 min | Ligation |
| 2 | 70℃ | 10 min | Inactivation |
| Store | 4℃ | Hold | Store |
Electrophoresis
Mimata
pHIL253, HD-5 on pSB1C3, Crp4 on pSB1C3
| Gel Concentration | Voltage | Time | Buffer |
|---|---|---|---|
| 1% | 100 V | 60 min | 1/2 x TBE |
Gel Extract
Mimata
pHIL253
FastGeneTM Gel/PCR Extraction kit (Nippon Genetics Co., Ltd)
DNA extraction from gel
Ligation
Mimata
pSB1C3 / oriAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence
| Reagent | Volume |
|---|---|
| pSB1C3 | 10 µL |
| oriAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence | 20 µL |
| Mighty Mix | 30 µL |
| Total | 60 µL |
Ligation
| Step | Temp. | Time | Process |
|---|---|---|---|
| 1 | 16℃ | 30 min | Ligation |
| 2 | 70℃ | 10 min | Inactivation |
| Store | 4℃ | Hold | Store |
Digestion
Sakai
XbaⅠ - oripAMβ1 - repE - Erythromycin resistant gene - SpeⅠ
| Reagent | Volume |
|---|---|
| XbaⅠ - oripAMβ1 - repE - Erythromycin resistant gene - SpeⅠ | 30 µL |
| XbaⅠ | 2 µL |
| SpeⅠ | 2 µL |
| 10 x M Buffer | 4 µL |
| DW | 2 µL |
| Total | 40 µL |
Digestion
| Step | Temp. | Time | Process |
|---|---|---|---|
| 1 | 37℃ | 120 min | Digestion |
| 2 | 80℃ | 20 min | Inactivation |
| Store | 4℃ | Hold | Store |
2015/09/06
Colony PCR
Fujita
Erythromycin resistant gene - BBa_B0015 on pSB1C3, Pcasei - BBa_B0034 - Sec signal sequence - Crp4 - BBa_B0034 - Sec signal sequence - BBa_E0040 - BBa_B0015 on pSB1C3, Pcasei - BBa_B0034 - Sec signal sequence - HD5 - BBa_B0034 - Sec signal sequence - BBa_E0040 - BBa_B0015
| Reagent | Volume |
|---|---|
| Single Colony | - |
| 100UP - EX - F 10 µM | 0.4 µL |
| 200DN - PS - R 10 µM | 0.4 µL |
| KAPA Taq | 5.0 µL |
| DW | 4.2 µL |
| Total | 10 µL |
3 Step Cycle (Tm value ≤ 63℃)
| Step | Temp. | Time | Process | Cycle |
|---|---|---|---|---|
| Start | 95℃ | 120 sec | Initialization | |
| Cycle 1 | 95℃ | 30 sec | Denaturation | 35 cycle |
| Cycle 2 | 57.6℃ | 30 sec | Annealing | 35 cycle |
| Cycle 3 | 72℃ | 210 sec | Elongation | 35 cycle |
| Finish | 72℃ | 120 sec | Final Elongation | |
| Store | 4℃ | Hold | Store |
Electrophoresis
Fujita
Erythromycin resistant gene - BBa_B0015 on pSB1C3, Pcasei - BBa_B0034 - Sec signal sequence - Crp4 - BBa_B0034 - Sec signal sequence - BBa_E0040 - BBa_B0015 on pSB1C3, Pcasei - BBa_B0034 - Sec signal sequence - HD5 - BBa_B0034 - Sec signal sequence - BBa_E0040 - BBa_B0015 (Colony PCR product)
| Gel Concentration | Voltage | Time | Buffer |
|---|---|---|---|
| 1% | 100 V | 30 min | 1/2 x TBE |
2015/09/07
Transformation
Sakai
oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence on pSB1C3
- Added 1.0 µL of oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence on pSB1C3 to 50 µL of thawed competent cells (JM1O9) on ice.
- Incubated on ice for 30 min.
- Heat-shocked for 60 sec at 42℃.
- Added 200 µL of LB.
- Incubated the cells for 2 hrs at 37℃.
- Spread 300 µL of the culture onto plate with LBC.
- Incubated the plate at 37℃ for 12 hrs.
