Team:HokkaidoU Japan/Notebook/plastic Week 12

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.

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.

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.

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.

=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!;)

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.

Negative control

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

Cultured for 48 hrs.