Team:HokkaidoU Japan/Notebook/plastic Week 12

From 2012.igem.org

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<div class="hokkaidou-notebook-daily">
<div class="hokkaidou-notebook-daily">
-
==September 17th==
+
===September 17th===
-
<div>
+
<div class="hokkaidou-section">
-
==Plasmid extraction==
+
====Plasmid extraction====
-
<p>
+
Plasmids of RBS-PhaC-RBS-PhaA-RBS-PhaB-dT on pSB1A2 and pTet(BBa_0040) on pSB1A2 were extracted.<br/>
Plasmids of RBS-PhaC-RBS-PhaA-RBS-PhaB-dT on pSB1A2 and pTet(BBa_0040) on pSB1A2 were extracted.<br/>
And then we got DNA solution of them.
And then we got DNA solution of them.
-
</p>
 
-
==Digestion==
+
====Digestion====
-
<p>
+
RBS-PhaC-RBS-PhaA-RBS-PhaB-dT on pSB1A2 was digested by XbaI and PstI.<br/>
-
RBS-PhaC-RBS-PhaA-RBS-PhaB-dT on pSB1A2 was digested with XbaI and PstI restriction sites.<br/>
+
pTet on pSB1A2 was also digested by SpeI and PstI.
-
pTet on pSB1A2 was also digested with SpeI and PstI.
+
-
</p>
+
-
==Gel extraction==
+
====Gel extraction====
-
<p>
+
We confirmed the succession of digestion by electrophoresis.<br/>
We confirmed the succession of digestion by electrophoresis.<br/>
And then DNA were extracted from TBE gel.
And then DNA were extracted from TBE gel.
-
</p>
 
-
==Ethanol precipitation==
+
====Ethanol precipitation====
-
<p>
+
The digested DNAs were condensed by Ethanol precipitation.
The digested DNAs were condensed by Ethanol precipitation.
-
</p>
 
-
==Ligation==
+
====Ligation====
-
<p>
+
RBS-PhaC-RBS-PhaA-RBS-PhaB-dT was ligated with pTet on pSB1A2.
RBS-PhaC-RBS-PhaA-RBS-PhaB-dT was ligated with pTet on pSB1A2.
-
</p>
 
-
==Transformation==
+
====Transformation====
-
<p>
+
The ligated DNA was transformed into E. coli (strain: JM109).<br/>
-
The ligated DNA was transformed into E.coli (strain: JM109).<br/>
+
And then we spread fungus liquid on LBA plates.
And then we spread fungus liquid on LBA plates.
-
</p>
 
 +
====Colony PCR====
 +
We confirmed the ligation of [RBS-B] and pSB1C3.
 +
The results showed that the ligation went well.
 +
 +
We chose three colonies and started the liquid culture.
 +
 +
====Liquid culture====
 +
The colonies of [RBS-phaC-RBS-phaA on pSB1A2] and [RBS-phaC-RBS-phaA-RBS-phaC-dT on pSB1A2] were condensed in LB.
 +
</div></div>
 +
 +
<div class="hokkaidou-notebook-daily">
 +
===September 18th===
 +
<div class="hokkaidou-section">
 +
====Colony PCR====
 +
We confirmed the succession of ligation RBS-PhaC-RBS-PhaA-RBS-PhaB-dT with pTet on pSB1A2 by colony PCR.<br/>
 +
RBS-PhaB-dT, a part of insert was multiplied.
 +
 +
====Sequencing====
 +
The sequence of RBS-PhaC-RBS-PhaA-RBS-PhaB-dT was analyzed.
 +
The result showed that...
 +
 +
====Plasmid extraction====
 +
Plasmids of [RBS-PhaC-RBS-PhaA on pSB1A2], [RBS-PhaB on pSB1C3] and [RBS-PhaC-RBS-PhaA-RBS-PhaB-dT on pSB1A2] were extracted.
 +
 +
====Digestion====
 +
[RBS-PhaC-RBS-PhaA on pSB1A2] was digested by EcoRI and SpeI restriction sites.<br/>
 +
[RBS-PhaB on pSB1C3] was digested by EcoRI and XbaI.<br>
 +
[RBS-PhaC-RBS-PhaA-RBS-PhaB-dT on pSB1A2] was digested by EcoRI and PstI.<br>
 +
[RBS-PhaB on pSB1C3] was digested by EcoRI and PstI.<br>
 +
But we failed the second digestion.
 +
 +
====Gel extraction====
 +
We confirmed succession of digestion by electrophoresis.
 +
And then DNA were extracted from TBE gel.
 +
 +
====Ethanol precipitation====
 +
The extracted DNAs were condensed by Ethanol precipitation.
 +
 +
====Ligation====
 +
[RBS-PhaC-RBS-PhaA-RBS-PhaB-dT] and pSB1C3 were ligated.
 +
 +
====Liquid culture====
 +
We started to incubate the colony of [RBS-PhaC-RBS-PhaA on pSB1A2] at 37C, 180 rpm to digest and ligate again.
 +
</div></div>
 +
 +
<div class="hokkaidou-notebook-daily">
 +
===September 19th===
 +
<div class="hokkaidou-section">
 +
====Plasmid extraction====
 +
Plasmids of [RBS-PhaC-RBS-PhaA on pSB1A2] and [pTet-RBS-PhaC-RBS-PhaA-RBS-PhaC-dT on pSB1A2] were extracted.
 +
 +
====Digestion====
 +
[RBS-PhaC-RBS-PhaA on pSB1A2] was digested by EcoRI and SpeI restriction sites.<br/>
 +
[RBS-PhaB on pSB1C3] was digested by EcoRI and SpeI.<br>
 +
[pTet-RBS-PhaC-RBS-PhaA-RBS-PhaC-dT on pSB1A2] was digested by EcoRI and PstI.<br>
 +
[RBS-PhaB on pSB1C3] was digested by EcoRI and PstI.<br>
 +
 +
====DNA extraction====
 +
The digested DNA were extracted.
 +
 +
====Ethanol precipitation====
 +
The extracted DNA were condensed by EtOH precipitation.
 +
 +
====Ligation====
 +
[RBS-PhaC-RBS-PhaA] and [RBS-PhaB on pSB1C3] were ligated.<br>
 +
[pTet-RBS-PhaC-RBS-PhaA-RBS-PhaB-dT] and pSB1C3 were ligated too.
 +
 +
====Transformation====
 +
The ligated DNA were transformed into E. coli (strain: JM109).<br/>
 +
E. coli solution was spread on LBC.
 +
 +
====Colony PCR====
 +
We confirmed the ligation [RBS-PhaC-RBS-PhaA-RBS-PhaB-dT] with [pSB1C3] by colony PCR.<br/>
 +
The results showed that the ligation went well.
 +
 +
We chose three colonies and started the liquid culture.
 +
</div></div>
 +
 +
<div class="hokkaidou-notebook-daily">
 +
===September 20th===
 +
<div class="hokkaidou-section">
 +
====Colony PCR====
 +
We confirmed the ligation [RBS-PhaC-RBS-PhaA] with [RBS-PhaB on pSB1C3] and
 +
[pTet-RBS-PhaC-RBS-PhaA-RBS-PhaB-dT] with [pSB1C3] by colony PCR.<br/>
 +
The results showed that the ligation went well.
 +
 +
We chose three colonies and started the liquid culture.
 +
</div></div>
 +
 +
<div class="hokkaidou-notebook-daily">
 +
===September 21st===
 +
<div class="hokkaidou-section">
 +
 +
===Plasmid extraction===
 +
Plasmids of [RBS-PhaC-RBS-PhaA-RBS-PhaB on pSB1C3] and [pTet-RBS-PhaC-RBS-PhaA-RBS-PhaC-dT on pSB1C3] were extracted.
 +
 +
We submitted our parts and finished sending them !
 +
Hooray!;)
 +
</div></div>
 +
 +
<div class="hokkaidou-notebook-daily">
 +
 +
===September 22nd===
 +
<div class="hokkaidou-section">
 +
====Liquid culture in large scale====
 +
We made cells which contains both plasmids of phaCAB on pGEM and Ag43 on pSTV28.
 +
Ab43 are induced by arabinose, and the production of P3HB is induced by glucose.
 +
 +
{|class="hokkaidou-table-digestion"
 +
|-
 +
|LB
 +
|mess up to 50 ml
 +
|-
 +
|50% Glucose
 +
|2 ml
 +
|-
 +
|Arabinose
 +
|2.5 ml
 +
|-
 +
|Amp(100 mg/ml)
 +
|50 ul
 +
|-
 +
|Cp
 +
|50 ul
 +
|-
 +
|}
 +
 +
Negative control
 +
#Gulcose (-)
 +
#Arabinose (-)
 +
 +
Cultured for 48 hrs.
 +
</div></div>
 +
 +
<div class="hokkaidou-notebook-daily">
 +
 +
===September 23rd===
 +
<div class="hokkaidou-section">
 +
====Transformation====
 +
Transformed BBa_R0011 for ligation of promoter with IPTG induction.
 +
 +
Incubated in LB+K plate.
</div></div>
</div></div>

Latest revision as of 22:11, 26 September 2012

Contents

September 17th

Plasmid extraction

Plasmids of RBS-PhaC-RBS-PhaA-RBS-PhaB-dT on pSB1A2 and pTet(BBa_0040) on pSB1A2 were extracted.
And then we got DNA solution of them.

Digestion

RBS-PhaC-RBS-PhaA-RBS-PhaB-dT on pSB1A2 was digested by XbaI and PstI.
pTet on pSB1A2 was also digested by SpeI and PstI.

Gel extraction

We confirmed the succession of digestion by electrophoresis.
And then DNA were extracted from TBE gel.

Ethanol precipitation

The digested DNAs were condensed by Ethanol precipitation.

Ligation

RBS-PhaC-RBS-PhaA-RBS-PhaB-dT was ligated with pTet on pSB1A2.

Transformation

The ligated DNA was transformed into E. coli (strain: JM109).
And then we spread fungus liquid on LBA plates.

Colony PCR

We confirmed the ligation of [RBS-B] and pSB1C3. The results showed that the ligation went well.

We chose three colonies and started the liquid culture.

Liquid culture

The colonies of [RBS-phaC-RBS-phaA on pSB1A2] and [RBS-phaC-RBS-phaA-RBS-phaC-dT on pSB1A2] were condensed in LB.

September 18th

Colony PCR

We confirmed the succession of ligation RBS-PhaC-RBS-PhaA-RBS-PhaB-dT with pTet on pSB1A2 by colony PCR.
RBS-PhaB-dT, a part of insert was multiplied.

Sequencing

The sequence of RBS-PhaC-RBS-PhaA-RBS-PhaB-dT was analyzed. The result showed that...

Plasmid extraction

Plasmids of [RBS-PhaC-RBS-PhaA on pSB1A2], [RBS-PhaB on pSB1C3] and [RBS-PhaC-RBS-PhaA-RBS-PhaB-dT on pSB1A2] were extracted.

Digestion

[RBS-PhaC-RBS-PhaA on pSB1A2] was digested by EcoRI and SpeI restriction sites.
[RBS-PhaB on pSB1C3] was digested by EcoRI and XbaI.
[RBS-PhaC-RBS-PhaA-RBS-PhaB-dT on pSB1A2] was digested by EcoRI and PstI.
[RBS-PhaB on pSB1C3] was digested by EcoRI and PstI.
But we failed the second digestion.

Gel extraction

We confirmed succession of digestion by electrophoresis. And then DNA were extracted from TBE gel.

Ethanol precipitation

The extracted DNAs were condensed by Ethanol precipitation.

Ligation

[RBS-PhaC-RBS-PhaA-RBS-PhaB-dT] and pSB1C3 were ligated.

Liquid culture

We started to incubate the colony of [RBS-PhaC-RBS-PhaA on pSB1A2] at 37C, 180 rpm to digest and ligate again.

September 19th

Plasmid extraction

Plasmids of [RBS-PhaC-RBS-PhaA on pSB1A2] and [pTet-RBS-PhaC-RBS-PhaA-RBS-PhaC-dT on pSB1A2] were extracted.

Digestion

[RBS-PhaC-RBS-PhaA on pSB1A2] was digested by EcoRI and SpeI restriction sites.
[RBS-PhaB on pSB1C3] was digested by EcoRI and SpeI.
[pTet-RBS-PhaC-RBS-PhaA-RBS-PhaC-dT on pSB1A2] was digested by EcoRI and PstI.
[RBS-PhaB on pSB1C3] was digested by EcoRI and PstI.

DNA extraction

The digested DNA were extracted.

Ethanol precipitation

The extracted DNA were condensed by EtOH precipitation.

Ligation

[RBS-PhaC-RBS-PhaA] and [RBS-PhaB on pSB1C3] were ligated.
[pTet-RBS-PhaC-RBS-PhaA-RBS-PhaB-dT] and pSB1C3 were ligated too.

Transformation

The ligated DNA were transformed into E. coli (strain: JM109).
E. coli solution was spread on LBC.

Colony PCR

We confirmed the ligation [RBS-PhaC-RBS-PhaA-RBS-PhaB-dT] with [pSB1C3] by colony PCR.
The results showed that the ligation went well.

We chose three colonies and started the liquid culture.

September 20th

Colony PCR

We confirmed the ligation [RBS-PhaC-RBS-PhaA] with [RBS-PhaB on pSB1C3] and [pTet-RBS-PhaC-RBS-PhaA-RBS-PhaB-dT] with [pSB1C3] by colony PCR.
The results showed that the ligation went well.

We chose three colonies and started the liquid culture.

September 21st

Plasmid extraction

Plasmids of [RBS-PhaC-RBS-PhaA-RBS-PhaB on pSB1C3] and [pTet-RBS-PhaC-RBS-PhaA-RBS-PhaC-dT on pSB1C3] were extracted.

We submitted our parts and finished sending them ! Hooray!;)

September 22nd

Liquid culture in large scale

We made cells which contains both plasmids of phaCAB on pGEM and Ag43 on pSTV28. Ab43 are induced by arabinose, and the production of P3HB is induced by glucose.

LB mess up to 50 ml
50% Glucose 2 ml
Arabinose 2.5 ml
Amp(100 mg/ml) 50 ul
Cp 50 ul

Negative control

  1. Gulcose (-)
  2. Arabinose (-)

Cultured for 48 hrs.

September 23rd

Transformation

Transformed BBa_R0011 for ligation of promoter with IPTG induction.

Incubated in LB+K plate.


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