Team:KAIST Korea/Notebook Labnote/2012 7

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KAIST Korea 2012 iGEM

Notebook : Labnote-July

Labnote

July

July 1st 2012

Moth 0109, 1202 TOPO cloning colony PCR
Results

0701Fig1

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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July 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

0703Fig1

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

0705Fig1

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

0705Fig2

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

0705Fig3

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

0705Fig4

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

0708Fig1

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.

0710Fig1

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

0710Fig2

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

0710Fig3

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

Induction of Moth_1202, 2312 and 2314 gene (sampling) and running SDS-PAGE gel.
We induced Moth_1202, 2312 and 2314 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.



Moth_0109 transformed cell confirm PCR – second trial
Results

0713Fig1

There was no band appeared on the gel, which means that we don’t have any colony with Moth_0109 gene.
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July 14th 2012

piBR181 vector single cut
piBR181 vector was cut with RE to check the size of this vector.

Results

0714Fig1



Moth_1202-pTrcHis2Aexpression check (Result)
Results

0714Fig2

Discussion

For each samples of different induction conditions, we run SDS-PAGE gel and checked the presence of 81.72kDa protein, bifunctional acetyl-CoA decarbonylase/synthase complex subunit alpha/beta. Although the band appeared slightly between 75kDa and 90kDa, we were not certain that this band is from Moth 1202 gene expression. This band also exists on the negative sample. Therefore, we concluded that expression of 1202 gene on pTrcHis2A vector has failed.


Moth_2312-pBAD/TOPO expression check (Result)
Results

0714Fig3

Discussion

For each samples of different induction conditions, we run SDS-PAGE gel and checked the presence of 98.60kDa protein formate dehydrogenase subunit alpha. Although the band significantly appeared slightly above 42kDa, this was not the correct size of protein. Therefore, we concluded that expression of 2312 gene on pBAD/TOPO vector has failed.


Moth_2314-pBAD/TOPO expression check (Result)
Results

0714Fig4

Discussion

For each samples of different induction conditions, we run SDS-PAGE gel and checked the presence of 78.07kDa protein formate dehydrogenase beta subunit. The band was not certain on the gel image. Therefore, we concluded that expression of 2314 gene on pBAD/TOPO vector has failed.


Moth_2314- pTrcHis2A expression check (Result)
Results

0714Fig5

Discussion

For each samples of different induction conditions, we run SDS-PAGE gel and checked the presence of 78.07kDa protein formate dehydrogenase beta subunit. The band was not certain on the gel image. Therefore, we concluded that expression of 2314 gene on pTrcHis2A vector has failed.


pre-culture of Moth_1202, 1198 and 0109 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 15th 2012

Induction of Moth_1202, 1198 and 0109 gene (sampling) and running SDS-PAGE gel.
We induced Moth-1202, 1198 and 0109 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.


 Flip Flop

Project design
Results

2_0715Fig1

The att B and att P sequences are recognition site of bacteriophage Bxb1 integrase. To prove our concept, we designed two plasmid called pPoC (proof of concept plasmid) and pPoCpi (proof of concept plasmid, promoter inverted). The pPoC insert and pPoCpi insert are cloned into pSB1C3 to become pPoC and pPoCpi.

The three sequences - Bacteriophage Bxb1 integrase, sequences between BBa_E1010 and BBa_E0040 of the inserts, pPoC insert and pPoCpi insert – are synthesized through gene synthesis service provided by BIONEER.

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July 16th 2012

Moth_1202-pBAD/TOPO expression check (Result)
Results

0716Fig1

Discussion

For each samples of different induction conditions, we run SDS-PAGE gel and checked the presence of 81.72kDa protein, bifunctional acetyl-CoA decarbonylase/synthase complex subunit alpha/beta. Although the band appeared slightly above 75kDa, we were not certain that this band is from Moth 1202 gene expression. We thought that band of size 81.72kDa should be more biased towards 90kDa band. Therefore, we concluded that expression of 1202 gene on pBAD vector has failed.


Moth_1198-pBAD/TOPO expression check (Result)
Results

0716Fig2

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 significantly appeared slightly above 42kDa. This means that protein has N terminal tag, of which the size is 13kDa.

35.08kDa + N-term Thioredoxin 13kDa = 48.08kDa

Therefore, the product protein locates slightly above 42kDa ladder



Moth_1198-pTrcHis2A expression check (Result)
Results

0716Fig3

Discussion

For each samples of different induction conditions, we run SDS-PAGE gel and checked the presence of 81.72kDa protein, bifunctional acetyl-CoA decarbonylase/synthase complex subunit alpha/beta. Although the band appeared slightly above 75kDa, we were not certain that this band is from Moth 1202 gene expression. We thought that band of size 81.72kDa should be more biased towards 90kDa band. Therefore, we concluded that expression of 1202 gene on pBAD vector has failed.


Moth_0109-pTrcHis2A expression check (Result)
Results

0716Fig4

Discussion

For each samples of different induction conditions, we run SDS-PAGE gel and checked the presence of 59.94kDa protein, formate--tetrahydrofolate ligase. The band is not significant, but appeared slightly above 54kDa marker in the 37℃ sample.
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July 17th 2012

 Flip Flop

Part preparation
We selected some parts, needed for this project, from the partregistry. We requested 10 parts which are not included in distribution kit.

Results

2_0717Fig1

Also, we designed primers for constructing ‘proof of concept plasmid’.

2_0717Fig2



Transformation of parts into TOP10
Parts:
  • BBa_E0040 (wild-type GFP); AmpR
  • BBa_E1010 (mRFP); KanR
Competent cell: TOP10
Method: heat-shock


Results

BBa_E0040: Colony formed, will be checked by restriction cut of purified plasmid DNA.
BBa_E1010: No colony


Discussions

BBa_E1010 will be transformed into MG1655 by electro-transformation to make sure.
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July 18th 2012

 Flip Flop

PCR amplification of parts
Parts:
  • BBa_E0040(720bp) with primer GFP-F and Plasmid1-R: 740bp
  • BBa_E1010(681bp) with primer Plasmid1-F and mRFP-R: 701bp
Template: distributed parts 1uL


Results

Gel electrophoresis

2_0718Fig1

PCR purification:

BBa_E0040: 48.2ng/uL (purity 1.88)
BBa_E1010: 104.3ng/uL (purity 1.88)


Discussions

The bands of both parts showed right size and yield is considerable.

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July 19th 2012

No Special Event!
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July 20th 2012

No Special Event!
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July 21st 2012

No Special Event!
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July 22nd 2012

pre-culture of MG 1655 cell transformed with Moth 1202 gene inserted pTrcHis2A and pBAD/mycHisC vector.
We did pre-culture of both pTrcHis2A 1202 and pBAD/mycHisC transformed cell into 5ml LB and incubated on 37℃ for 16 hours.

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July 23rd 2012

Moth 0109 vector double cut check
Restriction and buffers are mixed in the ratio as shown below.

0723Fig1

Results

0723Fig2

We checked the size of each broken fragments of vector, and found each bands appear about 1.6kb. After checking correct size of fragment, we sent mini-prepped vectors for sequencing.


Induction of Moth_1202 gene (sampling) and running SDS-PAGE gel.
Last time we induced the samples in 0.5mM, 1mM IPTG and 10mM arabinose conditions, but there were no significant band on the gel. So we induced Moth_1202 gene in pTrcHis2A and pBAD vector with more larger concentration of IPTG and arabinose conditions: IPTG 2mM, 3mM, 4mM and Arabinose 10mM, 30mM, 40mM. 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 24th 2012

Expression check of Moth_1202 on pBAD vector (Result)
Results


For each samples of different induction conditions, we run SDS-PAGE gel and checked the presence of 81.72 kDa protein, acetyl-CoA decarbonylase/synthase complex subunit alpha/beta. The band appeared significantly on the correct size for pTrcHis2A vector lanes.

0724Fig1

0724Fig2

0724Fig3

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July 25th 2012

Sequencing result of Moth_0109
We analyzed sequencing result from Macrogen using Multialin program.

Results



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July 26th 2012

Vector mini-prep for all genes except 0109 and FDH.
Results

0726Fig1



colony PCR of Moth_0109 gene
Results

0726Fig2

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July 27th 2012

Primer design for Gibson assembly
Results

0727Fig1

According to the Gibson assembly kit from NEB,


PCR amplification of fragments for Gibson assembly
Results

0727Fig2

PCR amplification result was successful except for 1199, 1191 genes; bands appear at the correct size. Correct size for each fragments are as below.

1201 : 1.3 + 0.3 = 1.6kb
1203 :
1204
1197
1198
1199
1191
1516

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July 28th 2012

pre-culture of pTrcHis2A-Moth0109 vector inserted MG1655 cell.
We did pre-culture of pTrcHis2A 0109 transformed cell into 5ml LB and incubated on 37℃ for 16 hours.

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July 29th 2012

PCR amplification of pBAD - Moth_1191 & 1199
We amplified target genes using PCR. Total reaction volume was 50uL and the elongation time was about 1kb/min.

Results

0729Fig1

second trial of Moth_1199, 1191 amplification

This was the second trial of amplifying Moth 1199 and 1191, but we found no band on the gel. So, we decreased annealing temperature down to 51℃, but this time we also couldn’t find one.



pre-culture of cell for vector miniprep - Moth_1191 & 1199
As there might be errors in vector miniprep step, we decided to pre-culture the cell once more.



Induction of Moth_0109 gene (sampling) and running SDS-PAGE gel.
We induced Moth-0109 gene in pTrcHis2A vector with different IPTG conditions and temperature: 0.5mM, 1mM and 37℃, 30℃. And then, 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 30th 2012

Expression optimization of Moth_0109 gene on pTrcHis2A (Result)
For each samples of different induction conditions, we run SDS-PAGE gel and checked the presence of 59.94 kDa protein, formate tetrahydrofolate ligase.

Results

0730Fig1

Discussion



PCR amplification of Moth_1191, 1199 gene (3rd and 4th trial)
We did PCR amplification of Moth_1191 and 1199 gene with original TOPO primer and Gibson primer.

Results

0730Fig2

0730Fig3

We thought that there might be some problem with vector preparation, so we tried mini-prep once more, and we got no band. Also, we tried colony PCR of 1191 and 1199 gene with Gibson primers, which has failed.
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July 31st 2012

PCR amplification of Moth_1191, 1199 gene (5th trial)
We did PCR amplification of Moth_1191 and 1199 gene with original TOPO primer and Gibson primer.

Results

0731Fig1

We can see that only the samples with TOPO primers had the correct size of oligonucleotide. Therefore, there might be some problems with forward primers.


pre-culture of pBAD/mycHisC-Moth0109 vector inserted MG1655 cell.
We did pre-culture of pBAD/mycHisC 0109 transformed cell into 5ml LB and incubated on 37℃ for 16 hours.

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