Team:KAIST Korea/Notebook Labnote/2012 8

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<div class="note-title">pPoCpi colony PCR</div>
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<span id="little">Among colonies #1~10, four colonies were inoculated.
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#1, 3, 4, 7
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Revision as of 03:57, 27 September 2012

KAIST Korea 2012 iGEM

Notebook : Labnote-August

Labnote

August

August 1st 2012

 PACKMAN

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

0801Fig1

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


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.



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

0801Fig2

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

pPoC and pPoCpi OE PCR with changed primers
Results

2_0801Fig1

→ Gel extracted
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August 2nd 2012

 PACKMAN

Primer design for FDH derived from Candida boidinii mutant
Results

0802Fig1

Discussion

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


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

0802Fig2

Discussion

The band of PAGE gel was not certain, so we

 Flip Flop

pPoC and pPoCpi cloning - Digestion
Restriction enzyme digestion

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

  • 2_0802Fig1

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

  • 2_0802Fig2




Bxb1 integrase transformation
Synthesized gene arrived.
Transformed into DH5α 
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August 3rd 2012

 Flip Flop

Bxb1 integrase-DH5α streaking
Discussions

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


pPoC and pPoCpi cloning - Ligation
Ligation - 16℃ overnight

2_0803Fig3

Transformed into DH5α chemically competent cell.


BBa_J04450 transformation into DH5α
For pSB1C3 vector amplification
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August 4th 2012

 PACKMAN

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

0804Fig1


 Flip Flop

BBa_J04450 and Bxb1-pGEM colony picking and inoculation.
Made master plates and inoculated in 3mL of LB.
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August 5th 2012

No Special Event!
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August 6th 2012

 Flip Flop

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


Requested part arrived
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) and BBa_K398005(pSB1C3) transformation
BBa_K381001(GFP): for GFP control
BBa_K398005(pSB1C3): for pSB1C3 template

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August 7th 2012

 PACKMAN

FDH gene PCR amplification
Results

0807Fig1

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.
0807Fig2



 Flip Flop

BBa_J04450(pSB1C3) enzyme cut check(single cut with EcoRI)
Results

2_0807Fig1

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


pPoC and pPoCpi OE PCR product nested PCR
Results

2_0807Fig2

→ Gel extracted


BBa_K398005 Plasmid miniprep & single cut check with EcoRI
Results

2_0807Fig3



Overlapping PCR
pPoC insert

2_0807Fig4

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


pPoCpi insert

2_0807Fig4

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


Results

2_0807Fig5

→ Concentration after gel extraction was not sufficient for cloning



Overlapping PCR template preparation
2_0807Fig6

2_0807Fig7

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August 8th 2012

 PACKMAN

FDH gene and pTrcHis2A, pBAD/mycHisC vector enzyme digestion
Procedure

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

0808Fig1

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


pTrcHis2A, pBAD/myc vector dephosphorylation
Procedure

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

0808Fig2

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.


ligation of FDH gene with pTrcHis2A and pBAD/mycHisC vectors
Procedure

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

0808Fig3

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


Primer amplification of 1191 and 1199
Results

0808Fig4

Discussion



 Flip Flop

Overlapping PCR
pPoC insert

2_0808Fig2

pPoCpi insert

2_0808Fig3


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


Results

2_0808Fig1

→ Gel extraction


Overlapping PCR template preparation
Results

2_0808Fig4

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August 9th 2012

 PACKMAN

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

0809Fig1

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 cloning into pTrcHis2a and pBADmycHisC
Bxb1 integrase: 1515bp

Results

2_0809Fig1

→ Gel extracted


Extracted OE PCR product cloning into pSB1C3
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.

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August 10th 2012

 Flip Flop

Bxb1-pTrc, pBAD colony inoculation


Overlapping PCR
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

2_0810Fig1

Discussions

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


pPoC, pPoCpi insert cloning into pSB1C3
pSB1C3 was derived from BBa_K398005

  1. Digestion – 37℃ 1hr, 80℃ 20min
  2. 2_0810Fig5

  3. Dephosphorylation – 37℃ 30min, 80℃ 10min
  4. 2_0810Fig6

  5. Ligation – 37℃ 2hr
  6. 2_0810Fig7



Ligated products were transformed into DH5α.



Transformed cell inoculation and colony PCR
Results

2_0810Fig8

Discussions

only pPoCpi construct cloning was completed 
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August 11th 2012

 PACKMAN

pTrcHis2A vector transformation into E.coli MG1655 cell
Procedure



pBAD/mycHisC-FDH transformation into TOP10


 Flip Flop

Bxb1 pTrcHis2A, pBADmycHisC plasmid DNA mini-prep
Results

single cut check

2_0811Fig1



pPoC and pPoCpi nested PCR
Results

nested PCR to prepare more insert DNA

2_0811Fig2


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August 12th 2012

 PACKMAN

colony PCR check of pTrcHis2A inserted FDH gene
Results

0812Fig1

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.


pBAD FDH MG1655 electroporation
Results

Fail
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August 13th 2012

 PACKMAN

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




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.


 Flip Flop

pPoCpi cloning into pSB1C3
Cloning was done by remaining DNA samples.

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August 14th 2012

 PACKMAN

colony PCR check of pBADmycHisC inserted FDH gene
Results

0814Fig1

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.


Expression check of Formate dehydrogenase on pTrcHis2A vector (Result)
Results

0814Fig2

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.


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. )


Primer design for the insertion of 1202 and 0109
Results

0814Fig3

Discussion

To eliminate uncertain spacer between genes, we inserted

 Flip Flop

Bxb1 pTrcHis2A and pPoCpi ligated products transformation
The products were transformed into TOP10

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August 15th 2012

 PACKMAN

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.

 Flip Flop

pPoCpi colony PCR
Results

2_08##Fig#

Among colonies #1~10, four colonies were inoculated. #1, 3, 4, 7
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August 16th 2012

 PACKMAN

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

0816Fig1

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.


0816Fig2



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.

 Flip Flop

pPoC and pPoCpi electro-transformation into MG1655
Strain: MG1655

Plasmid source:

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


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August 17th 2012

 PACKMAN

Expression check of Formate dehydrogenase.
Results

0817Fig1

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.


PCR amplification – second trial
Results

0817Fig2

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

0817Fig3


 Flip Flop

MG1655-pPoC, MG1655-pPoCpi streaking
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-pTrcHis2A_Bxb1 expression check
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.

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August 18th 2012

 PACKMAN

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

0818Fig1

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.


1202 and 0109 forward primer design
We designed new forward primer hoping no problem with it.

Results


 Flip Flop

MG1655-pPoC, MG1655-pPoCpi colony inoculation
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 electro-transformation into MG1655-pPoC, MG1655-pPoCpi
pTrcHis2A_Bxb1 is electro-transformed into MG1655 containing pPoC and pPoCpi.

Plasmid source: TOP10-pTrcHis2A_Bxb1


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August 19th 2012

No Special Event!
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August 20th 2012

No Special Event!
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August 21st 2012

No Special Event!
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August 22nd 2012

 Flip Flop

Primer design for Bxb1 integrase cloning
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

2_0822Fig1

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August 23rd 2012

No Special Event!
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August 24th 2012

No Special Event!
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August 25th 2012

 Flip Flop

pPoC-Bxb1 double transformants inoculation for prep & PCR check
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.

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August 26th 2012


 Flip Flop

pPoC-Bxb1 double transformants plasmid DNA miniprep & PCR check
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

2_0826Fig1

Confirm PCR

2_0826Fig2


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.


Bxb1 PCR with new primers
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
  5. Step 2 to 4: 34 cycles
  6. 72℃ for 10min
  7. 12℃ forever


Results

2_0826Fig3

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.


pGEM-Bxb1 template vector gel electrophoresis
To check whether the undesired bands were template vector, we diluted template vector with same conditions of PCR, 50X, and electrophoresized.

Results

2_0826Fig4


Discussion

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


Insert DNA (Bxb1 integrase) and vector (pTrc and pBAD) cloning
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August 27th 2012


 Flip Flop

Bxb1-pTrc and Bxb1-pBAD electrotransformation into MG1655


Bxb1 PCR with changed conditions
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 PCR conditions #2
1) 95℃ for 3min 1) 95℃ for 3min
2) 95℃ for 30sec 2) 95℃ for 30sec
3) 56.5℃ for 30sec 3) 58℃ for 30sec
4) 72℃ for 1min 30sec
  (Step 2 to 4: 34 cycles)
4) 72℃ for 1min 30sec
  (Step 2 to 4: 34 cycles)
5) 72℃ for 10min 5) 72℃ for 10min
6) 12℃ forever 6) 12℃ forever

Results

2_0827Fig1


Discussions

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


Bxb1, with GTG as start codon, PCR product 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 GTG) and vector (pTrc and pBAD) 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

2_0827Fig2


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

2_0827Fig3


Ligation: 16℃ overnight

2_0827Fig4



Bxb1, pPoC double transformants colony PCR
Colony #1, 4, 7, 10, 13, 14, 16, 19 and 22 from master plate

Results

2_0827Fig5


Discussions

The gel showed that all the colonies have Bxb1-pTrc vector. And also they all have pPoC or pPoCpi, because they show color.
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August 28th 2012

 Flip Flop

Bxb1-GTG-pTrc and Bxb1-GTG-pBAD electrotransformation into MG1655


Bxb1-pTrc and Bxb1-pBAD transformants check
Results

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



Bxb1-pTrc and Bxb1-pBAD transformants colony PCR
Results

2_0828Fig1





Bxb1-pTrc and Bxb1-pBAD transformants plasmid DNA miniprep
Results

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




mRFP, GFP 30mL culture and sampling
Results

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August 29th 2012


 Flip Flop

Bxb1 pBADmycHisC prepped plasmid DNA RE cut check
mini-prepped plasmid DNAs were cut with SnaBI

expected size : 5.6kb


Results

2_0829ig1


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August 30th 2012

 Flip Flop

Double transformants expression SDS-PAGE
Gel composition: 20%

Results

pPoC

2_0830Fig1

pPoCpo

2_0830Fig2

Results

We cannot see expression level change of fluorescent protein in SDS-PAGE.
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August 31st 2012

No Special Event!
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