Team:HokkaidoU Japan/Notebook/plastic Week 10

Single colony isolation=

The colony of pGEM PhaCAB in JM109 was isolated to plate1 and plate2.
The colony of pGEM PhaCAB in DH5α was isolated to plate3.

Plate 1~3 was started to incubate from 18:00.

Colony PCR

We confirmed the length of constructs, [PhaB on pSB1C3] and [RBS-PhaC-RBS-PhaA on pSB1A2] by colony PCR.
The result showed ligation to take PhaB on pSB1C3 went well.
But ligation [RBS-PhaC on pSB1A2] with [RBS-PhaA] was failed.

Liquid culture

We add 2 ml LB and 2 ul antibiotic to ideal colony suspension and begun to incubate at 37C, 180 rpm.

Colony PCR result

Colony PCR

We confirmed the length of constructs, [RBS-PhaA on pSB1A2], [RBS-PhaC on pSB1A2] and [PhaB on pSB1C3] by colony PCR.
Ideal plasmids were liquid cultured and spreaded on LBplates.

PCR

PhaB on pSB1C3 dimer was multiplied by PCR.
And then we got DNA solution of PhaB has prefix and suffix.

Plasmid extraction

We extracted the plasmids of [RBS-phaC] on pSB1A2 and [RBS-phaA] on pSB1A2 which was incubated from September 3rd. Then we got 50 ul of DNA solutions.

Colony PCR

We confirmed the length of constructs, [RBS-PhaC-RBS-PhaA on pSB1A2] by colony PCR.
The result showed some of ligation [RBS-PhaC on pSB1A2] with [RBS-PhaA] went well.

Gel extraction

Gel Extraction for digestion products. We used FastGene Gel&PCR extraction kit (NipponGenetics). Got 50 ul of DNA solution of phaB.

Liquid culture

The media of pGEM phaCAB was removed to polymer producing media.

Plasmid extracton

The plasmids, [RBS-PhaC-RBS-PhaA] were extracted.
And then we got 50ul DNA solution.

Sequence

We analyzed nucleotide sequence of construct made ever.
The result showed that PhaA sequence contained a mutation.
We decided to make construct including PhaA.

harvesting

@Taguchi lab The weight of new centrifuge tubes were measured.

JM109

1. pantothenic acid (+) 1.5 ml W:1.461 (W=tube weight with nothing inside.)
2. pantothenic acid (-) 1.5 ml W:1.457
3. pantothenic acid (+) 0.7 ml W:1.576

DH5a

1. pantothenic acid (+) 1.5 ml W:1.461
2. pantothenic acid (-) 1.5 ml W:1.461
3. pantothenic acid (+) 0.7 ml W:1.460

The cells were centrifuged and freeze dried for two days.