Difference between revisions of "Team:HokkaidoU Japan/Notebook/lcasei"

Line 764: Line 764:
 
</table>
 
</table>
 
<!-- Electrophoresis END -->
 
<!-- Electrophoresis END -->
 +
 +
 +
 +
 +
 +
<p class="nyannyan1">2015/09/05</p>
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
<!-- Colony PCR 3STEP -->
 +
<p class="nyannyan2">Colony PCR</p>
 +
<p class="nyannyan4"><span class="kinyuu">Fujita, Mimata</span></p>
 +
<p class="nyannyan3"><span class="kinyuu">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</span>
 +
<table class="hyounyannyan">
 +
<tr><th>Reagent</th><th>Volume</th></tr>
 +
<tr><td>Single Colony</td><td>-</td></tr>
 +
<tr><td><span class="kinyuu">100UP - EX - F</span> 10 μM</td><td><span class="kinyuu">0.4</span> μL</td></tr>
 +
<tr><td><span class="kinyuu">200DN - PS - R</span> 10 μM</td><td><span class="kinyuu">0.4</span> μL</td></tr>
 +
<tr><td>KAPA Taq</td><td><span class="kinyuu">5.0</span> μL</td></tr>
 +
<tr><td>DW</td><td><span class="kinyuu">4.2</span> μL</td></tr>
 +
<tr><td><b>Total</b></td><td><b><span class="kinyuu">10</span> μL</b></td></tr>
 +
</table>
 +
<p class="nyannyan3">3 Step Cycle (Tm value &le; 63℃)</p>
 +
<table class="hyounyannyan">
 +
<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>Cycle 1</td><td>95℃</td><td>30 sec</td><td>Denaturation</td><td><span class="kinyuu">35</span> cycle</td></tr>
 +
<tr><td>Cycle 2</td><td><span class="kinyuu">57.6</span>℃</td><td>30 sec</td><td>Annealing</td><td><span class="kinyuu">35</span> cycle</td></tr>
 +
<tr><td>Cycle 3</td><td>72℃</td><td>210 sec</td><td>Elongation</td><td><span class="kinyuu">35</span> cycle</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>
 +
</table>
 +
<!-- Colony PCR 3STEP END -->
 +
 +
<!-- Electrophoresis -->
 +
<p class="nyannyan2">Electrophoresis</p>
 +
<p class="nyannyan4"><span class="kinyuu">Fujita, Mimata</span></p>
 +
<p class="nyannyan3"><span class="kinyuu"><p class="nyannyan3"><span class="kinyuu">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</span></p>
 +
<table class="hyounyannyan">
 +
<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr>
 +
<tr><td><span class="kinyuu">1</span>%</td><td><span class="kinyuu"100</span> V</td><td><span class="kinyuu">30</span> min</td><td>1/2 x TBE</td></tr>
 +
</table>
 +
<!-- Electrophoresis END -->
 +
 +
<!-- Mini-prep -->
 +
<p class="nyannyan2">Mini-prep</p>
 +
<p class="nyannyan4"><span class="kinyuu">Mimata</span></p>
 +
<p class="nyannyan3"><span class="kinyuu">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</span>
 +
<br>FastGene<sup>TM</sup> Plasmid mini kit (Nippon Genetics Co.,Ltd)
 +
<br><span class="kinyuu">standard protocol</span></p>
 +
<!-- Mini-prep END -->
 +
 +
 +
<!-- Ligation -->
 +
<p class="nyannyan2">Ligation</p>
 +
<p class="nyannyan4"><span class="kinyuu">Mimata</span></p>
 +
<p class="nyannyan3"><span class="kinyuu">pSB1C3</span> / <span class="kinyuu">P<sub>casei</sub> - BBa_B0034 - Sec signal sequence - Crp4 - BBa_B0034 - Sec signal sequence - BBa_E0040 - BBa_B0015</span></p>
 +
<table class="hyounyannyan">
 +
<tbody><tr><th>Reagent</th><th>Volume</th></tr>
 +
<tr><td><span class="kinyuu">pSB1C3</span></td><td><span class="kinyuu">2.5</span> μL</td></tr>
 +
<tr><td><span class="kinyuu">P<sub>casei</sub> - BBa_B0034 - Sec signal sequence - Crp4 - BBa_B0034 - Sec signal sequence - BBa_E0040 - BBa_B0015</span></td><td><span class="kinyuu">30</span> μL</td></tr>
 +
<tr><td>Mighty Mix</td><td><span class="kinyuu">32.5</span> μL</td></tr>
 +
<tr><td><b>Total</b></td><td><b><span class="kinyuu">65</span> μL</b></td></tr>
 +
</tbody></table>
 +
<p class="nyannyan3">Ligation</p>
 +
<table class="hyounyannyan">
 +
<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>2</td><td>70℃</td><td>10 min</td><td>Inactivation</td></tr>
 +
<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td></tr>
 +
</tbody></table>
 +
<!-- Ligation END -->
 +
 +
<!-- Electrophoresis -->
 +
<p class="nyannyan2">Electrophoresis</p>
 +
<p class="nyannyan4"><span class="kinyuu">Mimata</span></p>
 +
<p class="nyannyan3"><span class="kinyuu">pHIL253, HD-5 on pSB1C3, Crp4 on pSB1C3</span></p>
 +
<table class="hyounyannyan">
 +
<tbody><tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr>
 +
<tr><td><span class="kinyuu">1</span>%</td><td><span class="kinyuu">100</span> V</td><td><span class="kinyuu">60</span> min</td><td>1/2 x TBE</td></tr>
 +
</tbody></table>
 +
<!-- Electrophoresis END -->
 +
 +
<!-- Gel Extract -->
 +
<p class="nyannyan2">Gel Extract</p>
 +
<p class="nyannyan4"><span class="kinyuu">Mimata</span></p>
 +
<p class="nyannyan3"><span class="kinyuu"><span class="kinyuu">pHIL253</span></span>
 +
<br>FastGene<sup>TM</sup> Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd)
 +
<br>DNA extraction from gel</p>
 +
<!-- Gel Extract END -->
 +
 +
 +
<!-- Ligation -->
 +
<p class="nyannyan2">Ligation</p>
 +
<p class="nyannyan4"><span class="kinyuu">Mimata</span></p>
 +
<p class="nyannyan3"><span class="kinyuu">pSB1C3</span> / <span class="kinyuu">oriAMβ1 - repE - Erythromycin resistant gene - P <sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence</span></p>
 +
<table class="hyounyannyan">
 +
<tbody><tr><th>Reagent</th><th>Volume</th></tr>
 +
<tr><td><span class="kinyuu">pSB1C3</span></td><td><span class="kinyuu">10</span> μL</td></tr>
 +
<tr><td><span class="kinyuu">oriAMβ1 - repE - Erythromycin resistant gene - P <sub>casei</sub> - RBS<sub>casei</sub> - Sec signal sequence</span></td><td><span class="kinyuu">20</span> μL</td></tr>
 +
<tr><td>Mighty Mix</td><td><span class="kinyuu">30</span> μL</td></tr>
 +
<tr><td><b>Total</b></td><td><b><span class="kinyuu">60</span> μL</b></td></tr>
 +
</tbody></table>
 +
<p class="nyannyan3">Ligation</p>
 +
<table class="hyounyannyan">
 +
<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>2</td><td>70℃</td><td>10 min</td><td>Inactivation</td></tr>
 +
<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td></tr>
 +
</tbody></table>
 +
<!-- Ligation END -->
 +
 +
<!-- Digestion -->
 +
<p class="nyannyan2">Digestion</p>
 +
<p class="nyannyan4"><span class="kinyuu">Sakai</span></p>
 +
<p class="nyannyan3"><span class="kinyuu">XbaⅠ - oripAMβ1 - repE - Erythromycin resistant gene - SpeⅠ</span></p>
 +
<table class="hyounyannyan">
 +
<tr><th>Reagent</th><th>Volume</th></tr>
 +
<tr><td><span class="kinyuu">XbaⅠ - oripAMβ1 - repE - Erythromycin resistant gene - SpeⅠ</span></td><td>30 μL</td></tr>
 +
<tr><td><span class="kinyuu">XbaⅠ</span></td><td>2 μL</td></tr>
 +
<tr><td><span class="kinyuu">SpeⅠ</span></td><td>2 μL</td></tr>
 +
<tr><td><span class="kinyuu">10 x M Buffer</span></td><td>4 μL</td></tr>
 +
        <tr><td><span class="kinyuu">DW</span></td><td>2 μL</td></tr>
 +
<tr><td><b>Total</b></td><td><b>40 μL</b></td></tr>
 +
</table>
 +
<p class="nyannyan3">Digestion</p>
 +
<table class="hyounyannyan">
 +
<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>2</td><td>80℃</td><td>20 min</td><td>Inactivation</td></tr>
 +
<tr><td>Store</td><td>4℃</td><td>Hold</td><td>Store</td></tr>
 +
</table>
 +
<!-- Digestion END -->
 +
 +
 +
<p class="nyannyan1">2015/09/06</p>
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
<!-- Colony PCR 3STEP -->
 +
<p class="nyannyan2">Colony PCR</p>
 +
<p class="nyannyan4"><span class="kinyuu">Fujita</span></p>
 +
<p class="nyannyan3"><span class="kinyuu">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</span></p>
 +
<table class="hyounyannyan">
 +
<tr><th>Reagent</th><th>Volume</th></tr>
 +
<tr><td>Single Colony</td><td>-</td></tr>
 +
<tr><td><span class="kinyuu">100UP - EX - F</span> 10 μM</td><td><span class="kinyuu">0.4</span> μL</td></tr>
 +
<tr><td><span class="kinyuu">200DN - PS - R</span> 10 μM</td><td><span class="kinyuu">0.4</span> μL</td></tr>
 +
<tr><td>KAPA Taq</td><td><span class="kinyuu">5.0</span> μL</td></tr>
 +
<tr><td>DW</td><td><span class="kinyuu">4.2</span> μL</td></tr>
 +
<tr><td><b>Total</b></td><td><b><span class="kinyuu">10</span> μL</b></td></tr>
 +
</table>
 +
<p class="nyannyan3">3 Step Cycle (Tm value &le; 63℃)</p>
 +
<table class="hyounyannyan">
 +
<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>Cycle 1</td><td>95℃</td><td>30 sec</td><td>Denaturation</td><td><span class="kinyuu">35</span> cycle</td></tr>
 +
<tr><td>Cycle 2</td><td><span class="kinyuu">57.6</span>℃</td><td>30 sec</td><td>Annealing</td><td><span class="kinyuu">35</span> cycle</td></tr>
 +
<tr><td>Cycle 3</td><td>72℃</td><td>210 sec</td><td>Elongation</td><td><span class="kinyuu">35</span> cycle</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>
 +
</table>
 +
<!-- Colony PCR 3STEP END -->
 +
 +
 +
<!-- Electrophoresis -->
 +
<p class="nyannyan2">Electrophoresis</p>
 +
<p class="nyannyan4"><span class="kinyuu">Fujita</span></p>
 +
<p class="nyannyan3"><span class="kinyuu">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 (Colony PCR product)</span></p>
 +
<table class="hyounyannyan">
 +
<tr><th>Gel Concentration</th><th>Voltage</th><th>Time</th><th>Buffer</th></tr>
 +
<tr><td><span class="kinyuu">1</span>%</td><td><span class="kinyuu">100</span> V</td><td><span class="kinyuu">30</span> min</td><td>1/2 x TBE</td></tr>
 +
</table>
 +
<!-- Electrophoresis END -->
 +
 +
 +
 +
 +
 +
 +
 +
  
  

Revision as of 22:20, 18 September 2015

Notebook

main1

L. casei

August

2015/08/11

Transformation

Mitsumoto, Onoda

pHIL253

  1. Added 5 μL of pHIL253 to μL of thawed competent cells (DH5α) on ice.
  2. Incubated on ice for 30 min.
  3. Heat-shocked for 60 sec at 42℃.
  4. Spread 300 μL of the culture onto plate with LBA.
  5. Incubated the plate at 37℃ for 16 hours.

2015/08/12

Liquid Culture

Mitsumoto

pHIL253

ReagentVolume
Single Colony-
LB2000 μL
Amp2 μL

Cultured for 20 hours.

2015/08/13

Mini-prep

Mitsumoto

pHIL253
FastGeneTM Plasmid mini kit (Nippon Genetics Co.,Ltd)
standard protocol

Preparation of Bacteria

Mitsumoto

AHU1910

  1. Added 5 mL of L. casei to MRS medium.
  2. Cultured overnight.
  3. Measured OD600 and diluted with MRS medium to set OD600 to 0.2.
  4. Stand cultured for 1.5 ~ 2 hours until OD600 is 0.4 ~ 0.5.
  5. Incubated the cells on ice for 10 min.
  6. Centrifuged at 6,000 g for 15 min at 4℃.
  7. Removed supernatant and added 30 mL of cooled PEB.
  8. Centrifuged at 6,000 g for 15 min at 4℃.
  9. Removed supernatant and added 30 mL of cooled PEB.
  10. Centrifuged at 6,000 g for 15 min at 4℃.
  11. Removed supernatant and added 1 mL of cooled PEB.

Electroporation

Mitsumoto

AHU1910

  1. Prepared the pHIL253 to 300 ng/10 μL (TE pH 8).
  2. Mixed 10 μL of plasmid and 200 μL of bacteria and incubated on ice for 10 min.
  3. Electoroporated at 2 kV with Gene Pulser (Bio-Rad) and Gene Pulser®/MicroPulserTM Electroporation Cuvettes(0.2 cm gap #1652086).
  4. Added 800 μL of cooled MRS medium and stand cultured for 3 hours in 30℃.
  5. Spread on MRS plate (Em 5 μg/mL),(Amp 5 μg/mL).

2015/08/19

Electrophoresis

Mitsumoto

pHIL253

Gel ConcentrationVoltageTimeBuffer
2%100V30 min1/2 x TBE

PCR

Mitsumoto

XbaⅠ - oripAMβ1 - repE - Erythromycin resistant gene - P casei - RBScasei - Sec signal sequence - SpeⅠ, proCrp4

ReagentVolume
pHIL2531 μL
XbaⅠ - oripAMβ1 - repE - F - primer 10 μM1 μL
SpeⅠ - Sec signal sequence - R - primer 10 μM1 μL
KOD - Plus - Neo1 μL
10 × PCR Buffer for KOD - Plus - Neo5 μL
2 mM dNTPs5 μL
25 mM MgSO43 μL
DW33 μL
Total50 μL

2 Step Cycle (Tm value ≥ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation35 cycle
Cycle 268℃120 secAnnealing / Elongation35 cycle
Store4℃HoldStore

2015/08/24

Ligation

Onoda

BBa_E0040 on pSB1A2 / XbaⅠ - oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence - SpeⅠ

ReagentVolume
BBa_E0040 on pSB1A21 μL
XbaⅠ - oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence - SpeⅠ2 μL
Mighty Mix3 μL
DW4 μL
Total10 μL

Ligation

StepTemp.TimeProcess
116℃30 minLigation
265℃10 minInactivation
Store4℃HoldStore

Ligation

Onoda

BBa_R0040 on pSB1C3 / XbaⅠ - oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence - SpeⅠ

ReagentVolume
BBa_R0040 on pSB1C31 μL
XbaⅠ - oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence - SpeⅠ2 μL
Mighty Mix3 μL
DW4 μL
Total10 μL

Ligation

StepTemp.TimeProcess
116℃30 minLigation
265℃10 minInactivation
Store4℃HoldStore

Ligation

Onoda

pSB1C3 XbaⅠ & SpeⅠ (Digestion product) / XbaⅠ - oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence - SpeⅠ

ReagentVolume
pSB1C3 XbaⅠ & SpeⅠ (Digestion product)1 μL
XbaⅠ - oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence - SpeⅠ2 μL
Mighty Mix3 μL
DW4 μL
Total10 μL

Ligation

StepTemp.TimeProcess
116℃30 minLigation
265℃10 minInactivation
Store4℃HoldStore

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

  1. 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.
  2. Incubated on ice for 30 min.
  3. Heat-shocked for 30 sec at 42℃.
  4. Added 200 μL of LB.
  5. Incubated the cells for 2 hrs at 37℃.
  6. Spread 300 μL of the culture onto plate with LBC.
  7. Incubated the plate at 37℃ for 18 hours.

2015/08/26

Preparation of Bacteria

Mimata, Sakai

AHU1910

  1. Added 5 mL of L. casei to MRS medium.
  2. Cultured overnight.
  3. Measured OD600 and diluted with MRS medium to set OD600 to 0.2.
  4. Stand cultured for 1.5 ~ 2 hours until OD600 is 0.4 ~ 0.5.
  5. Incubated the cells on ice for 10 min.
  6. Centrifuged at 6,000 g for 15 min at 4℃.
  7. Removed supernatant and added 30 mL of cooled PEB.
  8. Centrifuged at 6,000 g for 15 min at 4℃.
  9. Removed supernatant and added 30 mL of cooled PEB.
  10. Centrifuged at 6,000 g for 15 min at 4℃.
  11. Removed supernatant and added 1 mL of cooled PEB.

2015/08/27

Preparation of Bacteria

Mimata, Sakai

AHU1910

  1. Added 5 mL of L. casei to MRS medium.
  2. Cultured overnight.
  3. Measured OD600 and diluted with MRS medium to set OD600 to 0.2.
  4. Stand cultured for 1.5 ~ 2 hours until OD600 is 0.4 ~ 0.5.
  5. Incubated the cells on ice for 10 min.
  6. Centrifuged at 6,000 g for 15 min at 4℃.
  7. Removed supernatant and added 30 mL of cooled PEB.
  8. Centrifuged at 6,000 g for 15 min at 4℃.
  9. Removed supernatant and added 30 mL of cooled PEB.
  10. Centrifuged at 6,000 g for 15 min at 4℃.
  11. Removed supernatant and added 1 mL of cooled PEB.

Electroporation

Mimata, Sakai

AHU1910

  1. Prepared the plasmid to 300 ng/10 μL (TE pH 8).
  2. Mixed 10 μL of plasmid and 200 μL of bacteria and incubated on ice for 10 min.
  3. Electoroporated at 2 kV with Gene Pulser (Bio-Rad) and Gene Pulser®/MicroPulserTM Electroporation Cuvettes(0.2 cm gap #1652086).
  4. Added 800 μL of cooled MRS medium and stand cultured for 3 hours in 30℃.
  5. 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

ReagentVolume
Single Colony-
100UP - EX - F 10 μM0.4 μL
200DN - PS - R 10 μM0.4 μL
KAPA Taq5.0 μL
DW4.2 μL
Total10 μL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start95℃120 secInitialization
Cycle 195℃10 secDenaturation35 cycle
Cycle 257.6℃30 secAnnealing35 cycle
Cycle 368℃120 secElongation35 cycle
Store4℃HoldStore

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

ReagentVolume
Single Colony-
100UP - EX - F 10 μM0.4 μL
SpeⅠ - Sec signal sequence reverse 2 10 μM0.4 μL
KAPA Taq5.0 μL
DW4.2 μL
Total10 μL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start95℃120 secInitialization
Cycle 195℃10 secDenaturation35 cycle
Cycle 257.6℃30 secAnnealing35 cycle
Cycle 368℃120 secElongation35 cycle
Store4℃HoldStore

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 products), oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence BBa_E0040(colony PCR product)

Gel ConcentrationVoltageTimeBuffer
2%100V45 min1/2 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 ConcentrationVoltageTimeBuffer
1%100 V40 min1/2 x TBE

Dephosphorylation

Onoda

BBa_E0040 on pSB1A2, BBa_R0040 on pSB1C3, pSB1C3 XbaⅠ & SpeⅠ (Digestion product)

ReagentVolume
vector10 μL
Antarctic Phosphatase1 μL
Antarctic Phosphatase Buffer2 μL
DW7 μL
Total20 μL

Dephosphorylation

StepTemp.TimeProcess
137℃30 minDephosphorylation
265℃10 minInactivation
Store4℃HoldStore

Electrophoresis

Onoda

BBa_E0040 on pSB1A2, BBa_R0040 on pSB1C3, pSB1C3 XbaⅠ & SpeⅠ (Digestion product)

Gel ConcentrationVoltageTimeBuffer
1%100 V40 min1/2 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

ReagentVolume
oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence20 μL
XbaⅠ1 μL
SpeⅠ - HF1 μL
10 × M buffer3 μL
DW5 μL
Total30 μL

Digestion

StepTemp.TimeProcess
137℃120 minDigestion
280℃20 minInactivation
Store4℃HoldStore

Electrophoresis

Onoda

oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence

Gel ConcentrationVoltageTimeBuffer
1%100 V40 min1/2 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

ReagentVolume
BBa_E0040 on pSB1A2, BBa_R0040 on pSB1C33.5 μL
oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence8 μL
Mighty Mix11.5 μL
DW7 μL
Total30 μL

Ligation

StepTemp.TimeProcess
116℃30 minLigation
265℃10 minInactivation
Store4℃HoldStore

Ligation

Onoda

pSB1C3 XbaⅠ & SpeⅠ (Digestion product) / oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence

ReagentVolume
pSB1C3 XbaⅠ & SpeⅠ (Digestion product)20 μL
oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence3 μL
Mighty Mix23 μL
DW4 μL
Total50 μL

Ligation

StepTemp.TimeProcess
116℃30 minLigation
265℃10 minInactivation
Store4℃HoldStore

September

2015/09/01

Preparation of Bacteria

Mimata

AHU1910

  1. Added 5 mL of L. casei to MRS medium.
  2. Cultured overnight.
  3. Measured OD600 and diluted with MRS medium to set OD600 to 0.2.
  4. Stand cultured for 1.5 ~ 2 hours until OD600 is 0.4 ~ 0.5.
  5. Incubated the cells on ice for 10 min.
  6. Centrifuged at 6,000 g for 15 min at 4℃.
  7. Removed supernatant and added 30 mL of cooled PEB.
  8. Centrifuged at 6,000 g for 15 min at 4℃.
  9. Removed supernatant and added 30 mL of cooled PEB.
  10. Centrifuged at 6,000 g for 15 min at 4℃.
  11. Removed supernatant and added 1 mL of cooled PEB.

Electroporation

Mimata, Sakai

AHU1910

  1. Prepared the plasmid to 300 ng/10 μL (TE pH 8).
  2. Mixed 10 μL of plasmid and 200 μL of bacteria and incubated on ice for 10 min.
  3. Electoroporated at 2 kV with Gene Pulser (Bio-Rad) and Gene Pulser®/MicroPulserTM Electroporation Cuvettes(0.2 cm gap #1652086).
  4. Added 800 μL of cooled MRS medium and stand cultured for 3 hours in 30℃.
  5. 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 ConcentrationVoltageTimeBuffer
1%100 V40 min1/2 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,

  1. 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 (DH5a) on ice.
  2. Incubated on ice for 30 min.
  3. Heat-shocked for 30 sec at 42℃.
  4. Added 200 μL of LB.
  5. Incubated the cells for 2 hrs at 37℃.
  6. Spread 300 μL of the culture onto plate with LBCp.
  7. Incubated the plate at 37℃ for 2 hours.

Electrophoresis

Mitsumoto

EcoRⅠ - codon optimized - matured - HD-5 - PstⅠ

Gel ConcentrationVoltageTimeBuffer
1%100 V40 min1/2 x TBE

Electrophoresis

Mitsumoto

oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence

Gel ConcentrationVoltageTimeBuffer
1%100 V40 min1/2 x TBE

Electrophoresis

Mitsumoto

EcoRⅠ - oripAMB1 - repE - PstⅠ, EcoRⅠ - codon optimized - matured - HD-5 - PstⅠ

Gel ConcentrationVoltageTimeBuffer
1%100 V40 min1/2 x TBE

PCR

Ono

Erythromycin resistant gene, pAMβ1, HD-5, Crp4

ReagentVolume
Erythromycin resistant gene, oripAMβ1, HD-5, Crp41 μL
EX - F - Universal 10 μM1 μL
PS - R 10 μM1 μL
KOD - Plus - Neo1 μL
10x PCR Buffer for KOD - Plus - Neo5 μL
2 mM dNTPs5 μL
25 mM MgSO43 μL
DW33 μL
Total50 μL

2 Step Cycle (Tm value ≥ 63℃)

StepTemp.TimeProcessCycle
Start94℃120 secInitialization
Cycle 198℃10 secDenaturation30 cycle
Cycle 268℃30 secAnnealing / Elongation30 cycle
Store4℃HoldStore

Electrophoresis

Nishimura, Ono, Mimata

Crp4, HD-5

Gel ConcentrationVoltageTimeBuffer
1%100 V50 min1/2 x TBE

Digestion

Nisimura, Ono, Mimata

Erythromycin resistant gene

ReagentVolume
Erythromycin resistant gene60 μL
SpeⅠ2 μL
Total62 μL

Digestion

StepTemp.TimeProcess
137℃60 minDigestion
Store4℃HoldStore

Electrophoresis

Nishimura, Ono, Mimata

Erythromycin resistant gene

Gel ConcentrationVoltageTimeBuffer
2%100 V60 min1/2 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 ConcentrationVoltageTimeBuffer
1%100 V60 min1/2 x TBE

Electrophoresis

Nishimura

Erythromycin resistant gene, HD-5

Gel ConcentrationVoltageTimeBuffer
2%100 V60 min1/2 x TBE

Transformation

Fujita, Mimata

Erythromycin resistant gene - BBa_B0015, BBa_R0010 - BBa_B0034 - Crp4 - BBa_B0015, BBa_R0010 - BBa_B0034 - HD5 - BBa_B0015

  1. 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 (DH5a) on ice.
  2. Incubated on ice for 30 min.
  3. Heat-shocked for 30 sec at 42℃.
  4. Added 200 μL of LB.
  5. Incubated the cells for 2 hrs at 37℃.
  6. Spread 300 μL of the culture onto plate with LBC.
  7. Incubated the plate at 37℃ for 18 hours.

Digestion

Fujita, Mimata

oripAMβ1, BBa_R0010 - BBa_B0034 - HD5-BBa_B0015, BBa_R0010 - BBa_B0034 - Crp4 - BBa_B0015

ReagentVolume
oripAMβ1, BBa_R0010 - BBa_B0034 - HD5-BBa_B0015, BBa_R0010 - BBa_B0034 - Crp4 - BBa_B001520 μL
EcoRⅠ1 μL
PstⅠ1 μL
10 x H Buffer5 μL
DW23 μL
Total50 μL

Digestion

StepTemp.TimeProcess
137℃120 minDigestion
280℃20 minInactivation
Store4℃HoldStore

Digestion

Onoda

oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence

ReagentVolume
oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence20 μL
XbaⅠ1 μL
SpeⅠ1 μL
10 x M Buffer3 μL
DW5 μL
Total30 μL

Digestion

StepTemp.TimeProcess
137℃120 minDigestion
280℃20 minInactivation
Store4℃HoldStore

Electrophoresis

Onoda

oripAMβ1 - repE - Erythromycin resistant gene - Pcasei - RBScasei - Sec signal sequence (Digestion product)

Gel ConcentrationVoltageTimeBuffer
2%50 V60 min1/2 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

ReagentVolume
Single Colony-
100UP - EX - F 10 μM0.4 μL
200DN - PS - R 10 μM0.4 μL
KAPA Taq5.0 μL
DW4.2 μL
Total10 μL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start95℃120 secInitialization
Cycle 195℃30 secDenaturation35 cycle
Cycle 257.6℃30 secAnnealing35 cycle
Cycle 372℃210 secElongation35 cycle
Finish72℃120 secFinal Elongation
Store4℃HoldStore

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 ConcentrationVoltageTimeBuffer
1% V30 min1/2 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

pSB1C3 / Pcasei - BBa_B0034 - Sec signal sequence - Crp4 - BBa_B0034 - Sec signal sequence - BBa_E0040 - BBa_B0015

ReagentVolume
pSB1C32.5 μL
Pcasei - BBa_B0034 - Sec signal sequence - Crp4 - BBa_B0034 - Sec signal sequence - BBa_E0040 - BBa_B001530 μL
Mighty Mix32.5 μL
Total65 μL

Ligation

StepTemp.TimeProcess
116℃30 minLigation
270℃10 minInactivation
Store4℃HoldStore

Electrophoresis

Mimata

pHIL253, HD-5 on pSB1C3, Crp4 on pSB1C3

Gel ConcentrationVoltageTimeBuffer
1%100 V60 min1/2 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 - P casei - RBScasei - Sec signal sequence

ReagentVolume
pSB1C310 μL
oriAMβ1 - repE - Erythromycin resistant gene - P casei - RBScasei - Sec signal sequence20 μL
Mighty Mix30 μL
Total60 μL

Ligation

StepTemp.TimeProcess
116℃30 minLigation
270℃10 minInactivation
Store4℃HoldStore

Digestion

Sakai

XbaⅠ - oripAMβ1 - repE - Erythromycin resistant gene - SpeⅠ

ReagentVolume
XbaⅠ - oripAMβ1 - repE - Erythromycin resistant gene - SpeⅠ30 μL
XbaⅠ2 μL
SpeⅠ2 μL
10 x M Buffer4 μL
DW2 μL
Total40 μL

Digestion

StepTemp.TimeProcess
137℃120 minDigestion
280℃20 minInactivation
Store4℃HoldStore

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

ReagentVolume
Single Colony-
100UP - EX - F 10 μM0.4 μL
200DN - PS - R 10 μM0.4 μL
KAPA Taq5.0 μL
DW4.2 μL
Total10 μL

3 Step Cycle (Tm value ≤ 63℃)

StepTemp.TimeProcessCycle
Start95℃120 secInitialization
Cycle 195℃30 secDenaturation35 cycle
Cycle 257.6℃30 secAnnealing35 cycle
Cycle 372℃210 secElongation35 cycle
Finish72℃120 secFinal Elongation
Store4℃HoldStore

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 ConcentrationVoltageTimeBuffer
1%100 V30 min1/2 x TBE

←Notebook Top

Link to iGEM HokkaidoU website main1 Link to iGEM HokkaidoU twitter
Link to iGEM HokkaidoU facebook page main5