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August 1^st 2012

PCR amplification of Moth_1191, 1199 gene : decreasing annealing temperature down to 45℃

To make sure there’s no problem with unspecific binding or annealing temperature, we decreased annealing condition down to 45℃.

Results

There was no band and it became certain that there are no problems with annealing procedure.

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Induction of Moth_0109 gene (sampling) and running SDS-PAGE gel.

We induced Moth_0109 gene in pBAD/myc vector with different Arabinose conditions and temperature: 10mM, 30mM 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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PCR amplification of Moth_1191, 1199 gene with Gibson and TOPO primer

To see whether there are any problem with Gibson primers that we’ve designed, we did one more pcr amplification. Primers for Gibson assembly and TOPO cloning are mixed, and we could see any problems with forward or reverse primers.

Results

Each lane indicates:

• Moth_1191_1 : TOPO forward primer and Gibson reverse primer
• Moth_1191_2 : Gibson forward primer and TOPO reverse primer
• Moth_1199_1 : Gibson forward primer and TOPO reverse primer
• Moth_1199_2 : TOPO forward primer and Gibson reverse primer

Correct size of band appeared when topo forward primer and Gibson reverse primer was used in amplification of both genes. Therefore, we can conclude that there is some unknown problem with forward primers.

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August 2^nd 2012

Primer design for FDH derived from Candida boidinii mutant

Results

Discussion

These primers are universial primer that can be used for insertion of this gene into both pTrcHis2A vector and pBAD/mycHisC vector.

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Expression check of Moth_0109 gene on pBAD/mycHisC vector (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

Discussion

The band of PAGE gel was not certain, so we

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August 3^rd 2012

No Special Event!

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August 4^th 2012

1191 and 1199 forward primer design

We designed new forward primers for both 1191 and 1199 gene that are thought to solve problems appeared during PCR amplification.

Results

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August 5^th 2012

No Special Event!

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August 6^th 2012

No Special Event!

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August 7^th 2012

FDH gene PCR amplification

Results

Discussion

We amplified 2 samples of formate dehydrogenase gene with the primers we designed. From gel electrophoresis picture, we can say that oligonucleotides with the proper size 1100bp are present in the sample.

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August 8^th 2012

FDH gene and pTrcHis2A, pBAD/mycHisC vector enzyme digestion

Procedure

We mixed restriction enzymes and buffers with DNA samples as shown below.

After mixing the enzymes and buffer with the sample, we incubated the sample on 37℃ for 1 hour and then on 65℃ for 20minutes.

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pTrcHis2A, pBAD/myc vector dephosphorylation

Procedure

We mixed phosphatidase and buffer with DNA sample as shown below.

After mixing the enzyme and buffer with the sample, we incubated the sample on 37℃ for 1 hour 20minutes and then on 70℃ for 5minutes.

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ligation of FDH gene with pTrcHis2A and pBAD/mycHisC vectors

Procedure

We mixed ligase and buffer with DNA sample as shown below.

After mixing the enzyme and buffer with the sample, we incubated the sample on 16℃ for 16 hours.

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Primer amplification of 1191 and 1199

Results

Discussion

These

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August 9^th 2012

vector transformation into TOP10 cell

Transformation of pTrcHis2A vector into TOP10 cell was successful. So we did vector mini-preparation after the transformation. However, transformation of pBAD vector has failed, so we ligated the samples again.

Results

This is gel picture to confirm that FDH is well inserted into TOP10 cells. 3 wells on the left are PCR product of

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August 10^th 2012

No Special Event!

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August 11^th 2012

pTrcHis2A vector transformation into E.coli MG1655 cell

Procedure

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pBAD/mycHisC-FDH transformation into TOP10

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August 12^th 2012

colony PCR check of pTrcHis2A inserted FDH gene

Results

Discussion

We checked the presence of FDH gene by conducting colony PCR. Correct size of band appeared at about 1115bp, which means formate dehydrogenase is inserted correctly.

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pBAD FDH MG1655 electroporation

Results

Fail

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August 13^th 2012

FDH pBAD MG1655 electroporation

Procedure

We seeded cells on 7:10 AM and did cell-down on 10:00AM. And then we plated cells on 1:00PM and checked colony on 8/14 5:00AM

Results

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FDH pTrc MG1655 expression check (sampling and induction)

Procedure

We seeded cells (500λ of cells put into 50mL LB with 50λ Ampicillin) on 7 : 30 AM and checked Optical Density on 10:30pm. According to the O.D. value, we induced 37℃ sample on 10:30PM and did sampling on 4:30pm. Also, we induced 30℃ sample on 11:30pm and did sampling on 5:30pm. (6 hour induction) Each temperature samples were induced by different conditions ; 2mM and 3mM of IPTG.

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August 14^th 2012

colony PCR check of pBADmycHisC inserted FDH gene

Results

Discussion

We checked the presence of FDH gene by conducting colony PCR. Correct size of band appeared at about 1115 bp, which means formate dehydrogenase is inserted correctly.

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Expression check of Formate dehydrogenase on pTrcHis2A vector (Result)

Results

Discussion

For each samples of different induction conditions, we run SDS-PAGE gel and checked the presence of 43.7 kDa protein, formate dehydrogenase. The band appeared significantly on the correct size.

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FDH pBAD MG1655 seeding for expression check

Procedure

We seeded cell on 7:00PM, so we’ll incubate cell about 16hours. (This means we have to do next seeding on 11:00AM. )

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Primer design for the insertion of 1202 and 0109

Results

Discussion

To eliminate uncertain spacer between genes, we inserted

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August 15^th 2012

FDH pBAD MG1655 expression check

Procedure

We seeded cells on 11 : 30 AM and checked Optical Density on 1:30pm. According to the O.D. value, we induced 37℃ sample on 1:40pm and did sampling on 7:40pm. Also, we induced 30℃ sample on 3:30pm and did sampling on 9:30pm. (6 hour induction) Each temperature samples were induced by different conditions: 10mM of arabinose and 30mM of arabinose.

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August 16^th 2012

PCR amplification of 1201, 1202, 1203, 1198 and 1191

Results

Discussion

Primers arrived, and we began to do additional PCR amplification on the preparation for Gibson assembly. Each PCR sample has the correct size of oligonucleotides except Moth 1202 gene.

• 1191 : 921bp
  
• 1198 : 972bp + about 400bp = 1300 bp
  
• 1201 : 1341bp + ?
  
• 1202 : 2190bp +
  
• 1203 : 2025bp + 1600bp = 3625bp
  

We did PCR purification on the PCR product and the concentration/purity result was as shown below.

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Pre-culture of 0109

Discussion

There was no vector prepared for Moth 0109, so we did 5ml pre-culture on 10ml tube. Cell was grown about 16hours after pre-culture, and we did vector mini-prep on the sample. The concentration of the mini-prepped vector is 726.8ng/ul and the purity(A₂₆₀/A₂₈₀) was 1.86.

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August 17^th 2012

Expression check of Formate dehydrogenase.

Results

Discussion

For each samples of different induction conditions, we run SDS-PAGE gel and checked the presence of 40.xx kDa protein, formate dehydrogenase. The band appeared more significantly than the case of pTrcHis2A vector. Therefore, using pBAD promoter seems to produce more proteins.

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PCR amplification – second trial

Results

Discussion

As the band was not significant for 1203 and 1198 on previous trial and purity was low for 1191, we tried secondary PCR amplification on samples for Gibson assembly; 1202, 1203, 1198, 1191. Particularly for 1202 gene, we tried additional colony PCR, so that we could check any problem with vector mini-prep. (PCR solution for 1202 colony PCR was : 22ul

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August 18^th 2012

PCR amplification of 1202, 0109 gene with original primer and Gibson primers

Results

Each lane indicates:

• Moth_1202_1 : Gibson forward + original reverse
• Moth_1202_2 : Gibson reverse + original forward
• Moth_0109_1 : Gibson forward + original reverse
• Moth_0109_2 : Gibson reverse + original forward

There was correct size and unspecific size of band on 1202_1 sample. Although there are also unspecific band, 1202_2 sample has large band of correct size. 0109_1 sample has only unspecific band and 0109_2 sample has right size of band.

Discussion

When the PCR was unsuccessful, we first thought it might be problem of forward primer as in the case of 1199 and 1191. Our guess was correct, and we’re going to fix the problem with forward primers.

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1202 and 0109 forward primer design

We designed new forward primer hoping no problem with it.

Results

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August 19^th 2012

August 20^th 2012

August 21^st 2012

August 22^nd 2012

August 23^rd 2012

August 24^th 2012

August 25^th 2012

August 26^th 2012

August 27^th 2012

August 28^th 2012

August 29^th 2012

August 30^th 2012

August 31^st 2012