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Team:KAIST Korea/Notebook Labnote/2012 5
From 2012.igem.org
2012 KAIST Korea
Mail : kaist.igem.2012@gmail.com
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Notebook : Labnote-May
Labnote
MayMay 1st 2012
Transformation
With vector construct of Moth_1203, we transformed E. coli TOP10. For TOP10, we always used heat-shock method for transformation. In addition, today we transformed E. coli K-12 MG1655 strain with pTrcHis2A vectors carrying Moth_1197, 1198, 1199, 1201, 1204, 2314 genes. In this case, we used electroporation method for transformation.
Moth_0109, 1516, 1191 PCR
We studied genes involved in Wood-ljungdahl pathway in M. thermoacetica and we could find three genes relevant to the enzymes encoded by those genes of C. ljungdahlii; clju_37610, 37630, 37640, 37650. These genes are as follows; Moth_0109, 1191, 1516.
Results
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Back to the CalendarMay 2nd 2012
Vector construction with alternative genes
With PCR amplified genes (Moth_0109, 1191, 1516) and pTrcHis2A plasmid, we constructed vector for transformation into E. coli TOP10. We first examined the size of amplified genes and then digested them with restriction enzymes (HindIII and BamHI).
Results
Colony PCR: Evaluating transformation.
We performed colony PCR with transformed cells carrying Moth_1197, 1198, 1199, 1201, 1204, 2314 genes to check they were successfully transformed. As well as these transformed MG1655 strain samples, we also amplified Moth_1203 gene by colony PCR which is cloned into TOP10 strain.
Results
Moth_1201 and 1202 show bands of correct size. However we failed in Moth_1203 cloning.
All bands have appeared at correct position. However bands are not so clear that it is not easy to evaluate each gene. Hence, we did one more round of PCR for the genes of unclear bands.
We have got correctly sized bands of Moth_1204, 1197, 1198 and 1199. However Moth_1203 ended up with strange bands; both their size and pattern are strange.
Moth_2312 PCR with redesigned primer
Results
We got correctly sized band of Moth_2312 PCR amplified with redesigned primer. Now we know that the failure of cloning Moth_2312 was due to incorrectly designed primers.
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May 3rd 2012
Transformation: Moth_0109, 1191, 1516 into TOP10.
Results
We got bands of correct size after amplifying Moth_0109, 1191, 1516 by colony PCR. It means that we were successful in the cloning of these genes into TOP10 strain.
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May 4th 2012
Moth_2312 enzyme double-cut and ligation
Results
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May 5th 2012
No special event!
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May 6th 2012
No special event!
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May 7th 2012
Moth_0109, 1191, 1516 vector mini-prep for sequencing
Results
Moth_2312 colony PCR
Results
There was no band appeared on the gel. It seems that we failed in Moth_2312 cloning. We thought that the vector which is extracted from TOP10 strain can be the reason of this failure.
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May 8th 2012
Moth_2312 cloned TOP10 strain mini-prep
Results
Moth_2312 cloned vector single cut check with XhoI
Results
We got incorrect bands here. We totally failed the Moth_2312 gene cloning into TOP10.
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May 9th 2012
PCR amplification by redesigned primer: Moth_1203, 2312
Since both Moth_1203, 2312 gene were not successfully cloned into TOP10, we revised everything about these two genes. We guess that we might have missed some mistakes we’ve made when designing primers for these two genes. Thus, as we designed primers for Moth_2312 again a few days ago, we decided to do the same for Moth_1203. With newly synthesized primers, we PCR amplified both genes again for cloning.
Results
Moth_2312 also have been amplified and its size was examined. Afterwards, both genes were treated with restriction enzymes, XhoI and EcoRI, for cloning vector construction. Gel extracted results are as follows.
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- Left – PCR with new primer
- Right – gel extraction of PCR product
May 10th 2012
No special event!
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May 11th 2012
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May 12th 2012
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May 13th 2012
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May 14th 2012
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May 15th 2012
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May 16th 2012
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May 17th 2012
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May 18th 2012
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May 19th 2012
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May 20th 2012
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May 21st 2012
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May 22nd 2012
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May 23rd 2012
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May 24th 2012
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May 25th 2012
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May 26th 2012
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May 27th 2012
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May 28th 2012
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May 29th 2012
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May 30th 2012
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May 31st 2012
Arrival of primers
After cloning of most target genes into pTrcHis2A plasmid, we decided to use another kind of plasmid; TOPO vector. To do TOPO cloning, we had to design new primers for each gene and they have just arrived today. From now on, we will move on to next phase of our research, cloning Wood-ljungdahl pathway into TOPO vector and evaluate the expression.
