Team:HokkaidoU Japan/Notebook/plastic Week 12

From 2012.igem.org

(Difference between revisions)
Line 14: Line 14:
==Digestion==
==Digestion==
<p>
<p>
-
RBS-PhaC-RBS-PhaA-RBS-PhaB-dT on pSB1A2 was digested with XbaI and PstI.<br/>
+
RBS-PhaC-RBS-PhaA-RBS-PhaB-dT on pSB1A2 was digested by XbaI and PstI.<br/>
-
pTet on pSB1A2 was also digested with SpeI and PstI.
+
pTet on pSB1A2 was also digested by SpeI and PstI.
</p>
</p>
Line 36: Line 36:
==Transformation==
==Transformation==
<p>
<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.
Line 88: Line 88:
==Digestion==
==Digestion==
<p>
<p>
-
[RBS-PhaC-RBS-PhaA on pSB1A2] was digested with EcoRI and SpeI restriction sites.<br/>
+
[RBS-PhaC-RBS-PhaA on pSB1A2] was digested by EcoRI and SpeI restriction sites.<br/>
-
[RBS-PhaB on pSB1C3] was digested with EcoRI and XbaI.<br>
+
[RBS-PhaB on pSB1C3] was digested by EcoRI and XbaI.<br>
-
[RBS-PhaC-RBS-PhaA-RBS-PhaB-dT on pSB1A2] was digested with EcoRI and PstI.<br>
+
[RBS-PhaC-RBS-PhaA-RBS-PhaB-dT on pSB1A2] was digested by EcoRI and PstI.<br>
-
[RBS-PhaB on pSB1C3] was digested with EcoRI and PstI.<br>
+
[RBS-PhaB on pSB1C3] was digested by EcoRI and PstI.<br>
But we failed the second digestion.  
But we failed the second digestion.  
</p>
</p>
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==Liquid culture==
==Liquid culture==
<p>
<p>
-
We started to incubate the colony of [RBS-PhaC-RBS-PhaA on pSB1A2] at 37C, 180rpm to digest and ligate again.
+
We started to incubate the colony of [RBS-PhaC-RBS-PhaA on pSB1A2] at 37C, 180 rpm to digest and ligate again.
</p>
</p>
Line 139: Line 139:
==Digestion==
==Digestion==
<p>
<p>
-
[RBS-PhaC-RBS-PhaA on pSB1A2] was digested with EcoRI and SpeI restriction sites.<br/>
+
[RBS-PhaC-RBS-PhaA on pSB1A2] was digested by EcoRI and SpeI restriction sites.<br/>
-
[RBS-PhaB on pSB1C3] was digested with EcoRI and SpeI.<br>
+
[RBS-PhaB on pSB1C3] was digested by EcoRI and SpeI.<br>
-
[pTet-RBS-PhaC-RBS-PhaA-RBS-PhaC-dT on pSB1A2] was digested with EcoRI and PstI.<br>
+
[pTet-RBS-PhaC-RBS-PhaA-RBS-PhaC-dT on pSB1A2] was digested by EcoRI and PstI.<br>
-
[RBS-PhaB on pSB1C3] was digested with EcoRI and PstI.<br>
+
[RBS-PhaB on pSB1C3] was digested by EcoRI and PstI.<br>
</p>
</p>
==DNA extraction==
==DNA extraction==
<p>
<p>
-
The digested DNAs were extracted.
+
The digested DNA were extracted.
</p>
</p>
==Ethanol precipitation==
==Ethanol precipitation==
<p>
<p>
-
The extracted DNAs were condensed by EtOH precipitation.
+
The extracted DNA were condensed by EtOH precipitation.
</p>
</p>
Line 164: Line 164:
==Transformation==
==Transformation==
<p>
<p>
-
The ligated DNA were transformed into E.coli (strain: JM109).<br/>
+
The ligated DNA were transformed into E. coli (strain: JM109).<br/>
-
E.Coli solution was spread on LBC.
+
E. coli solution was spread on LBC.
</p>
</p>
Line 211: Line 211:
<div>
<div>
-
 
-
 
-
----------
 
==Plasmid extraction==
==Plasmid extraction==
<p>
<p>
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|2.5 ml
|2.5 ml
|-
|-
-
|Amp(100mg/ml)
+
|Amp(100 mg/ml)
|50 ul
|50 ul
|-
|-
Line 257: Line 254:
#Arabinose (-)
#Arabinose (-)
-
Cultured for 48hrs.
+
Cultured for 48 hrs.
</p>
</p>

Revision as of 17:41, 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 22th

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 23th

Transformation

Transformed BBa_R0011 for ligation of promoter with IPTG induction. Cultured in LB+K plate.