Team:HokkaidoU Japan/Notebook/plastic Week 11

PCR

PhaA was multiplied from pGEM with XbaI and SpeI restriction sites by PCR.

Digestion

PhaA was digested with XbaI and SpeI. RBS on pSB1A2 was digested with SpeI.

Gel extraction

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

Ligation

RBS on pSB1A2 was ligated with PhaA and PhaB.

Transformation

These ligated DNAs transformed into E.coli (strain: DH5α).
And then we spread fungus liquid added LB on plates.

Sequencing

We analyzed the sequence which contains phaA to check whether there are mutations.

Colony PCR

The colony of RBS+phaB on 1A2 was

Digestion

RBS-PhaB on pSB1A2 was digested with EcoRI and SpeI restriction sites and dT(B0015) on pSB1AK3 was also digested with EcoRI and XbaI.

Ligation

The fragment of RBS-PhaB and dT(B0015) on pSB1AK3 was ligated.
However, because of my stupid mistake, both fragment failed to ligate. I deeply feel sorry to Mr.Kawata and the E. coli.

Polymer extraction

@Taguchi lab
The dried cells were removed to glass test tubes. Weight of plastic tubes were measured in order to estimate how much the cells weighed.

1. pantothenic acid (+) 1.5 ml W:1.466 (W=tube weight with dried cells inside.)
2. pantothenic acid (-) 1.5 ml W:1.467
3. pantothenic acid (+) 0.7 ml W:1.461

DH5a

1. pantothenic acid (+) 1.5 ml W:1.468
2. pantothenic acid (-) 1.5 ml W:1.468
3. pantothenic acid (+) 0.7 ml W:1.459

We couldn't remove all the cells to the glass tubes because the cells remained on the plastic tubes walls. The weight of plastic tubes were measured again to estimate the amount of removed cells.

1. pantothenic acid (+) 1.5 ml W:1.461 (W=tube weight with nothing inside.)
2. pantothenic acid (-) 1.5 ml W:1.467
3. pantothenic acid (+) 0.7 ml W:1.458

DH5a

1. pantothenic acid (+) 1.5 ml W:1.462
2. pantothenic acid (-) 1.5 ml W:1.464
3. pantothenic acid (+) 0.7 ml W:1.459

From these data, we can estimate the weight of the dried cells.

1. pantothenic acid (+) 1.5 ml W:0.144 (W=weight of dried cells.)
2. pantothenic acid (-) 1.5 ml W:0.099
3. pantothenic acid (+) 0.7 ml W:0.039

DH5a

1. pantothenic acid (+) 1.5 ml W:0.007
2. pantothenic acid (-) 1.5 ml W:0.006
3. pantothenic acid (+) 0.7 ml W:-----

Chloroform was added to the dried cells and was incubated on 60C for 48 hrs.

Digestion

We were worried about yesterdaysãlab work, so we tried it again.
RBS-PhaB on pSB1A2 was digested with EcoRI and SpeI restriction sites and dT(B0015) on pSB1AK3 was also digested by EcoRI and XbaI.

Gel extraction

We confirmed the succession of digestion by electrophoresis, and extracted the DNAs from TBE gel.

Ethanol precipitation

The DNA solution of RBS-PhaB and dT on pSB1AK3 were concentrated by EtOH precipitation.

Ligation

RBS-PhaB was ligated with dT on pSB1AK3.
We added LB to fungus liquid mixed ligated DNAs and then spread it on LBK plates.

Digestion

To submit our construct as a BioBrick, we had to ligate our part samples in pSB1C3.
RBS-phaC on pSB1A2, RBS-phaA and pSB1C3 was digested by EcoRI and PstI.

Gel extraction

We confirmed the succession of digestion by electrophoresis, and extracted the DNAs from TBE gel.

Polymer extraction

The samples were filiterized and were removed to another test tubes. I made a 1.7 mL loss of sample2.

Colony PCR

We confirmed whether or not RBS-PhaB was ligated with dT on pSB1AK3 correctly by colony PCR.

Liquid culture

We added 2 ml LB with 2 ul kanamycin to pre-cultured colony suspension, and then started to incubate at 37C, 180 rpm.

Plasmid extraction

Plasmids of RBS-PhaB-dT on pSB1AK3 were extracted.
And then we got DNA solution of it.

Digestion

RBS-PhaB-dT on pSB1AK3 was digested with XbaI and PstI restriction sites.
RBS-PhaC-RBS-PhaA on pSB1A2 was also digested by SpeI and PstI.

Gel extraction

We confirmed succession of digestion by electrophoresis.
Digested DNAs were extracted from TBE gel.

Ethanol precipitation

The digested DNA were condensed by EtOH precipitation.

Ligation

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

Transformation

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

Gel extraction

The plasmids of [RBS-phaB on pSB1C3] and [RBS-phaC-RBS-phaA on pSB1C3] were extracted by electrophoresis. And then DNA were extracted from TBE gel.

Ethanol precipitation

[RBS-phaA] and [RBS-phaC] were condensed by Ethanol precipitation.

Ligation

[RBS-phaA] and [RBS-phaC] was ligated with pSB1C3.

Digestion

[RBS-phaB on pSB1A2] was digested by EcoRI and PstI.

Colony PCR

We confirmed the ligation of RBS-PhaB-dT and RBS-PhaC-RBS-PhaA on pSB1A2 by colony PCR.

Liquid culture

We added 2 ml LB and 2 ul antibiotic to pre-cultured colony suspension.
And then started to incubate at 37C, 180 rpm.
Bacteria hold BBa_J23109, BBa_J23114, BBa_J23101 and BBa_J23102, constitutive promoter each of them have different expression intensity and pTet(BBa_R0040) were also begun to incubate.

Colony PCR

We confirmed the ligation of [RBS-phaA on pSB1C3] and [RBS-phaC on pSB1C3] by colony PCR.

Ligation

[RBS-phaB] was ligated with pSB1C3.