PACKMAN

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.

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.

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.

　Flip Flop

Results


→ Gel extracted

　PACKMAN

Results


Discussion

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

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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Restriction enzyme digestion

• Enzyme pre-mix / each reaction – 37℃ 1hr, 80℃ 20min





• Digestion mixture – 37℃ 1hr, 80℃ 20min

Synthesized gene arrived.
Transformed into DH5α 

　Flip Flop

Discussions

Synthesized gene was cloned into pGEM-B1 vector. It seems the vector concentration is too high so that too much colonies may appeared.

Ligation - 16℃ overnight


Transformed into DH5α chemically competent cell.

For pSB1C3 vector amplification

　PACKMAN

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

Results

　Flip Flop

Made master plates and inoculated in 3mL of LB.

No Special Event!

　Flip Flop

BBa_J04450(pSB1C3): 285.8ng/uL
purity:2.10

Each parts were streaked on appropriate plates.

• BBa_I11020
• BBa_I11022
• BBa_I11023
• BBa_I11030
• BBa_K137007
• BBa_K137008
• BBa_K137010
• BBa_K112001
• BBa_K112141
• BBa_K112142

BBa_K381001(GFP): for GFP control
BBa_K398005(pSB1C3): for pSB1C3 template

　PACKMAN

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.

　Flip Flop

Results


Vector was double digested. The insert and vector sizes were correct

Results


→ Gel extracted

Results

pPoC insert

• HSTaq Pol MM : 25uL
• DW : 1uL
• templates : 22uL
• primers : 2uL

pPoCpi insert

• HSTaq Pol MM : 25uL
• templates : 23.2uL
• primers : 2uL

Results


→ Concentration after gel extraction was not sufficient for cloning

　PACKMAN

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.

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.

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.

Results


Discussion

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pPoC insert


pPoCpi insert

• HSTaq Pol MM : 25uL
• DW : 5.2uL
• templates : 19.8uL
• primers : 2uL
• pfu-x : 0.1uL

Results


→ Gel extraction

Results

　PACKMAN

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

　Flip Flop

Bxb1 integrase: 1515bp

Results


→ Gel extracted

pSB1C3 was originated from BBa_K398005.

Transformed into TOP10

Results

Transformed colony was grown in LB media. No colony shows fluorescence.

Discussions

We think cloning failed.

　Flip Flop

In this time, PCR was done first 18 cycles without any primer.
After that, we add 1uL of primers. And then PCR was done 20 cycles more.

Results


Discussions

With this OE PCR conditions, yield was better than previous conditions.

pSB1C3 was derived from BBa_K398005

1. Digestion – 37℃ 1hr, 80℃ 20min



2. Dephosphorylation – 37℃ 30min, 80℃ 10min



3. Ligation – 37℃ 2hr

Ligated products were transformed into DH5α.

Results


Discussions

only pPoCpi construct cloning was completed 

　PACKMAN

Procedure

　Flip Flop

Results

single cut check

Results

nested PCR to prepare more insert DNA

　PACKMAN

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.

Results

Fail

　PACKMAN

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

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.

　Flip Flop

Cloning was done by remaining DNA samples.

　PACKMAN

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.

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.

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. )

Results


Discussion

To eliminate uncertain spacer between genes, we inserted

　Flip Flop

The products were transformed into TOP10

　PACKMAN

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.

　Flip Flop

Results


Among colonies #1~10, four colonies were inoculated. #1, 3, 4, 7

Results


Among colonies #1~20, three colonies were inoculated. #2, 5, 19

　PACKMAN

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.

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.

　Flip Flop

Strain: MG1655

Plasmid source:

• pPoC: from DH5α-pPoC colony #1 and TOP10-pPoC colony #1
• pPoCpi: from TOP10-pPoC colony #1 and #3

　PACKMAN

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.

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

　Flip Flop

We could not distinguish single colony from plates. Therefore we scraped cells and streaked onto new plates.

Discussions

Wild-type E.coli showed more reddish and yellowish color than competent strains, DH5α and TOP10. The promoter BBa_J23119 may show different strength in-between strains.

TOP10 containing pTrcHis2A was cultured in 30mL of LB with 1% of Ampicillin.
IPTG induced when OD = 0.4
Sampling: 8 hours after induction, 1010 cells.
Only soluble fractions were PAGEed.

　PACKMAN

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.

We designed new forward primer hoping no problem with it.

Results


Newly designed primers contain overlapping region with former DNA fragments, artificial ribosome binding site, and overlapping region with 1202 and 0109 each.

　Flip Flop

Two colonies from each plate were inoculated into 3mL of LB containing 1% of chloramphenicol.

Discussions

In 12 hours, cell cultures show colors compared to another common E.coli culture. When they are centrifuged, pellet shows clear and definite yellow or red color.

pTrcHis2A_Bxb1 is electro-transformed into MG1655 containing pPoC and pPoCpi.

Plasmid source: TOP10-pTrcHis2A_Bxb1

No Special Event!

No Special Event!

No Special Event!

　Flip Flop

Primers for Bxb1 integrase cloning were designed. For controlling basal transcription level optimization, a mutant primer which has GTG rather than ATG for its start codon was also ordered.

Results

No Special Event!

　PACKMAN

Results



Discussion

PCR product of Moth 0109 gene and 1191 gene has run on the gel. There was only faint band of 0109 fragment but the product band of 1191 gene was significantly shown on the gel. After gel electrophoresis, we tried PCR of 0109 once more and got significant amount of fragments.

　Flip Flop

Double transformants 1 through 24 were inoculated into 3ml of LB containing 1% of Cm and Ap. For our convenience, three colonies were inoculated in one culture tube.

　Flip Flop

Inoculated cultures were mipi-preped. And Bxb1-pTrc plasmid was checked by PCR. In case of pPoC, it would be check by color.

Results

Vector


Confirm PCR



Discussions

In confirm PCR, all the colonies seem to have Bxb1 vector. Colony #1, 4, 7, 10, 13, 14, 16, 19 and 22 will be inoculated.

For cloning of Bxb1 into pBAD and mutating its start codon from ATG to GTG, we PCR pGEM-B1-Bxb1 vector as template. Samples were duplicated

PCR conditions

1. 95℃ for 3min
2. 95℃ for 30sec
3. 55℃ for 30sec
4. 72℃ for 1min 30sec
Step 2 to 4: 34 cycles
5. 72℃ for 10min
6. 12℃ forever

Results


Duplicated samples were purified in one DNA binding column.

ATG: 297.9ng/uL (purity: 1.90)
GTG: purify failed. Sample lost

Discussions

There were multiple bands in our PCR products. We will PCR again Bxb1-GTG with different conditions. Template vector may show larger bands.

To check whether the undesired bands were template vector, we diluted template vector with same conditions of PCR, 50X, and electrophoresized.

Results



Discussion

The band was different from undesired bands from Bxb1 ATG, GTG PCR products.

　Flip Flop

For cloning of Bxb1 into pBAD and mutating its start codon from ATG to GTG, we PCR pGEM-B1-Bxb1 vector as template. Samples were duplicated


Results



Discussions

Still, undesired bands appeared. We do not know where they come from. We decide to extract desired DNA with gel extraction.

PCR products were gel extracted.

Results

• Bxb1 GTG cond. #1 - #1: 128.2ng/uL (purity: 1.89)
• Bxb1 GTG cond. #1 - #2: 79.0ng/uL (purity: 1.89)
• Bxb1 GTG cond. #2 - #1: 110.7ng/uL (purity: 1.89)
• Bxb1 GTG cond. #2 - #2: 66.7ng/uL (purity: 1.94)

Discussions

Bxb1 GTG cond. #1 - #1 and Bxb1 GTG cond. #2 - #1 will be used to cloning.

Insert DNA: Bxb1 integrase mutated by primer.
Vectro DNA: pTrcHis2A, pBAD-mycHisC

Prepared DNA concentration:

• Bxb1 GTG cond. #1 - #1: 128.2ng/uL (purity: 1.89)
• Bxb1 GTG cond. #2 - #1: 110.7ng/uL (purity: 1.89)
• pTrcHis2A: 116.1ng/uL
• pBAD-mycHisC: 128.7ng/uL

Enzyme digestion: 37℃ for 1hr 30min, 80℃ for 20min



Dephosphorylation: 37℃ for 30min, 70℃ for 5min



Ligation: 16℃ overnight

Colony #1, 4, 7, 10, 13, 14, 16, 19 and 22 from master plate

Results



Discussions

The gel showed that all the colonies have Bxb1-pTrc vector. And also they all have pPoC or pPoCpi, because they show color.

　Flip Flop

Results

Bxb1-pTrc: No colony
Bxb1-pBAD: Colonies appeared and incoculated into 3mL of LB containing 1% of Ap

Results

Results

Colony #2: 109.8ng/uL, purity = 1.87
Colony #3: 138.9ng/uL, purity = 1.74

Results

　Flip Flop

mini-prepped plasmid DNAs were cut with SnaBI

expected size : 5.6kb

Results



　PACKMAN

Results



Discussion

PCR for Moth 1202 gene has failed. No band for Moth 1202 gene has appeared on the gel.

Results

Among three methods to assemble DNA fragments, we chose first method, which is assembly of PCR amplified DNA fragments.
0.2umoles of each fragments are added into the reaction sample, and volume to be added for each fragments has calculated on the assumption that average MW of each base is 660Da. Table below is the result of calculation.

Fragment 1

Fragment 2



Discussion

As we can only assemble the fragments through Gibson Assembly method and not amplify them, there would be very small amount of template remaining in the tube. We thought that’s why we couldn’t see any band on the gel. So we did PCR purification of the fragment once more and saw if the band appears on the gel.

　Flip Flop

Gel composition: 20%

Results

pPoC


pPoCpo


Results

We cannot see expression level change of fluorescent protein in SDS-PAGE.

No Special Event!