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Team:KAIST Korea/Notebook Labnote/2012 7
From 2012.igem.org
2012 KAIST Korea
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Notebook : Labnote-July
Labnote
JulyJuly 1st 2012
Moth 0109, 1202 TOPO cloning colony PCR
Results
We did colony PCR for the 5 colony of TOPO cloned samples, but there was no band, which means TOPO cloning has failed for these two genes.
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Back to the CalendarJuly 2nd 2012
fdnG deletion PCP20 curing
fdnG deletion PCP20 prep, single cut
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July 3rd 2012
Moth_0109, 1202 PCR
As TOPO cloning of Moth_0109 and 1202 gene failed, we ran one more PCR to prepare for TOPO cloning.
Results
For each of the two genes, correct size of oligonucleotide exists in the PCR product. We noticed that there is an other restriction enzyme site in the Moth 0109, 1202. It means that it’s impossible to reuse TOPO vector. We got other vector which has same pBAD promoter with TOPO cloning. Also we redesigned the primer for Moth 0109, 1202.
pre-culture of Moth_1197 and 1198 inserted MG1655 cell.
We did pre-culture of vector transformed cell into 3ml LB and incubated on 37℃ for 16 hours.
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July 4th 2012
Induction of Moth_1197, Moth_1198 gene (sampling) and running SDS-PAGE gel.
We induced Moth_1197 and Moth_1198 gene in pTrcHis2A and pBAD vector with various IPTG and L-arabinose conditions: IPTG 0.5mM, 1mM and Arabinose 10mM, 30mM (0.02% and 0.2%). Temperature conditions are : 37℃, 30℃. Cells were cultured anaerobically in the anaerobic chamber. After 6 hours of induction, we run the sample into SDS-PAGE gel. The gel is stained with coomassie blue for 1 hour and de-stained for overnight.
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July 5th 2012
fdhF Knockout PCR ( negative and knockout) and PCP20?
Results
Moth_1197-pBAD/TOPO expression check (Result)
Results
Discussion
For each samples of different induction conditions, we run SDS-PAGE gel and checked the presence of 28.61 kDa protein, Methyltetrahydrofolate : corrinoid/iron sulfur protein methyltransferase. The band significantly appeared slightly above 42kDa. This means that protein has N and C terminal tag, of which the size is 13kDa and 3kDa each.
28.61kDa + N-term Thioredoxin 13kDa + C-term V5, 6xHis 3kDa = 44.61kDa
Therefore, the product protein locates slightly above 42kDa ladder.
Moth_1197-pTrcHis2A expression check (Result)
Results
Discussion
For each samples of different induction conditions, we run SDS-PAGE gel and checked the presence of 28.61 kDa protein, Methyltetrahydrofolate : corrinoid/iron sulfur protein methyltransferase. The band significantly appeared between 24kDa and 35kDa. This means that protein has C terminal tag,
28.61kDa + 2.14kDa = 30.75kDa
The band that does not exist on negative sample is roughly on this size. Therefore, we can say that this protein is also expressed on pTrcHis2A vector. However, we decided to use pBAD/TOPO vector because it has expressed the protein more significantly.
Moth_1198-pBAD/TOPO expression check (Result)
Results
Discussion
For each samples of different induction conditions, we run SDS-PAGE gel and checked the presence of 35.08kDa protein acetyl-CoA decarbonylase/synthase complex subunit delta. But the band was not certain on the gel image.
Moth_1198-pTrcHis2A expression check (Result)
Results
Discussion
For each samples of different induction conditions, we run SDS-PAGE gel and checked the presence of 35.08kDa protein acetyl-CoA decarbonylase/synthase complex subunit delta. The band of correct size appeared on the soluble fraction of each sample.
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July 6th 2012
No Special Event!
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July 7th 2012
No Special Event!
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July 8th 2012
Moth 0109, 1202 colony PCR for TOPO cloning to TOP10
Results
1202 colony PCR, The size of bands are correct. We succeed Moth 0109, 1202 gene cloning.
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July 9th 2012
fdoG knockout confirm PCR
Results
Moth 0109 and Moth 1202 electro transformation into MG1655 cell
Procedure
We did mini-prep to get the vectors which has Moth 0109, 1202 each from TOP10. We did electroporation to MG1655.
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July 10th 2012
piBR181 vector arrived
piBR181 vector is known to accommodate oligonucleotide over 100kb. This vector was made from commercial pET-28a (+) vectors to accommodate standardized Biobricks parts. The work proceeded by designing a 181 bp long synthetic DNA fragment for efficient construction of an expression vector for monocistronic assembly of genes provided that each contains its own promoter, operator, ribosome-binding site and transcriptional terminator. A 181 bp long ds DNA construct was designed and ligated to pET-28a (+) (5369 bp) at BglII and XhoI restriction sites to generate piBR181 (5301 bp) expression vector replacing original bio-parts. The newly constructed vector contains SpeI-HindIII single cloning restriction site between RBS and TT sites however, BglII and BamHI/XhoI restriction sites are present before promoter and after TT sites for multi-monocistronic operon assembly.
We got this standardized vector and vector map from Professor Jae Kyung, Song and it arrived today.
▶▶Click to download Original document of piBR181 vector
Moth 0109 transformed cell confirm PCR
Results
There was no band appeared on the gel, which means that we don’t have any colony with Moth 0109 gene.
Moth 1202 transformed cell confirm PCR
Results
Band size is smaller than Moth 1202. It means we failed to transform Moth 1202 gene in to MG1655.
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July 11th 2012
No Special Event!
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July 12th 2012
pre-culture of Moth_1202, 2312 and 2315 inserted MG1655 cell.
We did pre-culture of vector transformed cell into 3ml LB and incubated on 37℃ for 16 hours.
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July 13th 2012
July 14th 2012
July 15th 2012
