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Team:KAIST Korea/Notebook Labnote/2012 8
From 2012.igem.org
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<span id="little">Among three methods to assemble DNA fragments, we chose first method, which is assembly of PCR amplified DNA fragments. </br> | <span id="little">Among three methods to assemble DNA fragments, we chose first method, which is assembly of PCR amplified DNA fragments. </br> | ||
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.</br> | 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.</br> | ||
| - | Fragment 1</br> | + | </br>Fragment 1</br> |
</span> | </span> | ||
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<div style="clear:both;"></div> | <div style="clear:both;"></div> | ||
| - | <span id="little">Fragment 2</br> | + | <span id="little"></br>Fragment 2</br> |
</span> | </span> | ||
Revision as of 01:54, 27 October 2012
2012 KAIST Korea
Mail : kaist.igem.2012@gmail.com
Twitter : twitter.com/KAIST_iGEM_2012
Facebook : www.facebook.com/KAISTiGEM2012
Notebook : Labnote-August
Labnote
AugustAugust 1st 2012
PACKMANPCR 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.
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
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
Flip FloppPoC and pPoCpi OE PCR with changed primers
Results
→ Gel extracted
Back to the Calendar
Back to the CalendarAugust 2nd 2012
PACKMANPrimer 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.
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
Flip FloppPoC and pPoCpi cloning - Digestion
Restriction enzyme digestion
- Enzyme pre-mix / each reaction – 37℃ 1hr, 80℃ 20min
- Digestion mixture – 37℃ 1hr, 80℃ 20min
Bxb1 integrase transformation
Synthesized gene arrived.
Transformed into DH5α
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August 3rd 2012
Flip FlopBxb1 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
Transformed into DH5α chemically competent cell.
BBa_J04450 transformation into DH5α
For pSB1C3 vector amplification
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August 4th 2012
PACKMAN1191 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
Flip FlopBBa_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 FlopBBa_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
PACKMANFDH 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.
Flip FlopBBa_J04450(pSB1C3) enzyme cut check(single cut with EcoRI)
Results
Vector was double digested. The insert and vector sizes were correct
pPoC and pPoCpi OE PCR product nested PCR
Results
→ Gel extracted
BBa_K398005 Plasmid miniprep & single cut check with EcoRI
Results
Overlapping PCR
pPoC insert
→ Concentration after gel extraction was not sufficient for cloning
- HSTaq Pol MM : 25uL
- DW : 1uL
- templates : 22uL
- primers : 2uL
- HSTaq Pol MM : 25uL
- templates : 23.2uL
- primers : 2uL
Overlapping PCR template preparation
August 8th 2012
PACKMANFDH 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.
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.
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.
Primer amplification of 1191 and 1199
Results
Discussion
Flip FlopOverlapping PCR
pPoC insert
pPoCpi insert
→ Gel extraction
- HSTaq Pol MM : 25uL
- DW : 5.2uL
- templates : 19.8uL
- primers : 2uL
- pfu-x : 0.1uL
Overlapping PCR template preparation
Results
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August 9th 2012
PACKMANvector 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
Flip FlopBxb1 integrase cloning into pTrcHis2a and pBADmycHisC
Bxb1 integrase: 1515bp
Results
→ 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
Discussions
With this OE PCR conditions, yield was better than previous conditions.
pPoC, pPoCpi insert cloning into pSB1C3
pSB1C3 was derived from BBa_K398005
- Digestion – 37℃ 1hr, 80℃ 20min
- Dephosphorylation – 37℃ 30min, 80℃ 10min
- Ligation – 37℃ 2hr
Transformed cell inoculation and colony PCR
Results
Discussions
only pPoCpi construct cloning was completed
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August 11th 2012
PACKMANpTrcHis2A vector transformation into E.coli MG1655 cell
Procedure
pBAD/mycHisC-FDH transformation into TOP10
Flip FlopBxb1 pTrcHis2A, pBADmycHisC plasmid DNA mini-prep
Results
single cut check
pPoC and pPoCpi nested PCR
Results
nested PCR to prepare more insert DNA
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August 12th 2012
PACKMANcolony 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.
pBAD FDH MG1655 electroporation
Results
Fail
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August 13th 2012
PACKMANFDH 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 FloppPoCpi cloning into pSB1C3
Cloning was done by remaining DNA samples.
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August 14th 2012
PACKMANcolony 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.
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.
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
Discussion
To eliminate uncertain spacer between genes, we inserted
Flip FlopBxb1 pTrcHis2A and pPoCpi ligated products transformation
The products were transformed into TOP10
Back to the Calendar
August 15th 2012
PACKMANFDH 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 FloppPoCpi colony PCR
Results
Among colonies #1~10, four colonies were inoculated.
#1, 3, 4, 7
Bxb1 pTrcHis2A colony PCR
Results
Among colonies #1~20, three colonies were inoculated.
#2, 5, 19
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August 16th 2012
PACKMANPCR 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
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 FloppPoC and pPoCpi electro-transformation into MG1655
Strain: MG1655
Plasmid source:
Back to the Calendar
- pPoC: from DH5α-pPoC colony #1 and TOP10-pPoC colony #1
- pPoCpi: from TOP10-pPoC colony #1 and #3
August 17th 2012
PACKMANExpression 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.
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
Flip FlopMG1655-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.
Back to the Calendar
August 18th 2012
PACKMANPCR 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
1202 and 0109 forward primer design
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 FlopMG1655-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!
Back to the Calendar
August 21st 2012
No Special Event!
Back to the Calendar
August 22nd 2012
Flip FlopPrimer 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
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August 23rd 2012
No Special Event!
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August 24th 2012
PACKMAN 0109, 1191 PCR with new primers
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.
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August 25th 2012
Flip FloppPoC-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 FloppPoC-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
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.
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
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.
- 95℃ for 3min
- 95℃ for 30sec
- 55℃ for 30sec
- 72℃ for 1min 30sec Step 2 to 4: 34 cycles
- 72℃ for 10min
- 12℃ forever
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
Discussion
The band was different from undesired bands from Bxb1 ATG, GTG PCR products.
Insert DNA (Bxb1 integrase) and vector (pTrc and pBAD) cloning
August 27th 2012
Flip FlopBxb1-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
Results
Discussions
Still, undesired bands appeared. We do not know where they come from. We decide to extract desired DNA with gel extraction.
| 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 |
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)
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:
Dephosphorylation: 37℃ for 30min, 70℃ for 5min
Ligation: 16℃ overnight
- 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
Bxb1, pPoC double transformants colony PCR
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.
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August 28th 2012
Flip FlopBxb1-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
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 FlopBxb1 pBADmycHisC prepped plasmid DNA RE cut check
mini-prepped plasmid DNAs were cut with SnaBI
expected size : 5.6kb
Results
PACKMAN
PACKMAN 1202 PCR to make fragments for Gibson assembly
Results
Discussion
PCR for Moth 1202 gene has failed. No band for Moth 1202 gene has appeared on the gel.
Gibson assembly – assembly of pcr fragments.
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.
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August 30th 2012
Flip FlopDouble transformants expression SDS-PAGE
Gel composition: 20%
Results
pPoC
pPoCpo
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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