Team:KAIST Korea/Notebook Labnote/2012 9

From 2012.igem.org

(Difference between revisions)
 
Line 1,186: Line 1,186:
<section id="27">
<section id="27">
<div class="date">September 27<sup>th</sup> 2012</div></br>
<div class="date">September 27<sup>th</sup> 2012</div></br>
-
<div id="content_note" >
+
<img style="height:3px;width:269px;margin:0px 5px 0px 0px; "src="http://2012.igem.org/wiki/images/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:35px;float:bottom;"id="packman" src='http://2012.igem.org/wiki/images/0/03/KAIST_PACKMAN.png'/> PACKMAN<img style="height:3px;width:262px; margin:0px 0px 0px 5px;"src="http://2012.igem.org/wiki/images/1/1b/KAIST_horizontal_gray.png"/></br>
-
<span id="little">No Special Event!</span>
+
<div class="note-title">Pre-culture of Gibson F1, F2 colonies</div>
-
</br>
+
 
 +
<div id="content_note" >
-
</div>
+
<b>Results</b></br></br>
 +
<span id="little">We checked that there were several colonies on the plate. 10 colonies for each fragment were picked, and culture into 3mL LB media containing 30ul Kanamycin.</br></br></span>
 +
</div>
<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="http://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="http://2012.igem.org/wiki/images/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="http://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="http://2012.igem.org/wiki/images/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
</section>
</section>
<section id="28">
<section id="28">
<div class="date">September 28<sup>th</sup> 2012</div></br>
<div class="date">September 28<sup>th</sup> 2012</div></br>
-
<div id="content_note" >
+
<img style="height:3px;width:269px;margin:0px 5px 0px 0px; "src="http://2012.igem.org/wiki/images/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:35px;float:bottom;"id="packman" src='http://2012.igem.org/wiki/images/0/03/KAIST_PACKMAN.png'/> PACKMAN<img style="height:3px;width:262px; margin:0px 0px 0px 5px;"src="http://2012.igem.org/wiki/images/1/1b/KAIST_horizontal_gray.png"/></br>
-
<span id="little">No Special Event!</span>
+
<div class="note-title">Vector preparation and PCR</div>
-
</br>
+
 
 +
<div id="content_note" >
-
</div>
+
<b>Results</b></br></br>
 +
<span id="little">For each cultured cells, vectors were mini-prepped, and pcr was done with proper primers (F1 : 1201 F, 1197R, F2 : 1198F, 1516R)</br></br></span>
 +
</div>
<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="http://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="http://2012.igem.org/wiki/images/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="http://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="http://2012.igem.org/wiki/images/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
</section>
</section>

Latest revision as of 03:59, 27 October 2012

KAIST Korea 2012 iGEM

Notebook : Labnote-September

Labnote

September

September 1st 2012

 PACKMAN
fdh pBAD, fdh pTrc colony PCR check
Results

0818Fig1


Discussion

To check the presence of FDH gene, we picked the colony from the plate and did colony PCR. For all the colonies we picked, we confirmed that correct size of FDH is present.
 Flip Flop

Bxb1_GTG_pBAD cloning with remaining DNAs
Remaining Bxb1_GTG insert was cloned into pBADmycHisC vector. And electrotransformed into MG1655.

Back to the Calendar
September 2nd 2012

 Flip Flop

Bxb1_ATG_pTrc, pBAD, Bxb1_GTG_pTrc miniprep for double transformation, Bxb1_GTG_pBAD colony inoculation and vector miniprep NcoI single cut check.
Results

2_0902Fig1

Discussions

All the vectors showed right sizes. But Bxb1_GTG_pBAD colony #1 through 3 have undesired bands. They may be the supercoiled vector.


Bxb1_ATG_pTrc, pBAD, Bxb1_GTG_pTrc double transformation into pPoC and pPoCpi
Back to the Calendar
September 3rd 2012

 PACKMAN
1201 1197 1198 1516 PCR for Gibson assembly.
Results

0818Fig1


Discussion

There was no band for 1516 gene. Band of all three genes appeared at the right size.


1516 PCR for Gibson assembly.
Results

0818Fig1


Discussion

Also for this time, there was no right size of band appeared for 1516 gene.

We,
1. changed the fraction of primers down to 0.5ul each, (lane 1)
2. changed the polymerase to pfu-x, (lane3)
3. used hybrid primers ( TOPO forward, Gibson reverse : lane 4 and TOPO reverse, Gibson forward : lane 5)
Lane 2 was the control experiment with Hotstartaq polymerase.



Pre-culture of piBR181 vector containing cells.
Procedure

We did 3ml pre-culture on five 10ml tubes for the cells containing each gene. Cell was grown about 16hours after pre-culture, and then induced with proper inducer.

 Flip Flop

Bxb1 double transformants vector miniprep & PCR check
Results

2_0903Fig1

Discussions

Seemed to every colonies have appropriate vectors.
Back to the Calendar
September 4th 2012

 PACKMAN
1516 PCR for Gibson assembly.
Results

0818Fig1


Discussion

Also for this time, there was no right size of band appeared for 1516 gene.
Back to the Calendar
September 5th 2012

 PACKMAN
1201 1197 1198 PCR amplification
Results

0818Fig1


Discussion

To amplify the amount of each fragment, we did PCR amplification of each gene once more. Correct size of bands appeared.


1197 1516 PCR amplification
Results

0818Fig1


Discussion

To amplify the amount of each fragments, we did PCR amplification of 1197 once more. And we did PCR amplification of 1516 gene once more with the same primers. Correct size of bands appeared for 1197 gene, but there was no band for 1516 gene. .

Back to the Calendar
September 6th 2012

 PACKMAN
1198 1516PCR amplification
Results

0818Fig1


Discussion

To amplify the amount of each fragments, we did PCR amplification of 1198 once more. And we did PCR amplification of 1516 gene once more with the same primers. For 1516 gene, we couldn’t have right size of band for new primers. So we decided to use the fragments of 1516 that does not contain terminator sequence.
Back to the Calendar
September 7th 2012

 PACKMAN
Pre-culture of 0109, 1191, FDH containing cells.
Discussion

We did 5ml pre-culture on 10ml tube for the cells containing each gene. Cell was grown about 16hours after pre-culture, and then induced with proper inducer.

Back to the Calendar
September 8th 2012

 PACKMAN
FDH pTrc & pBAD/0109 pBAD/1191 pBAD MG1655 expression check (sampling and induction)
Procedure

We seeded cells (500λ of cells put into 50mL LB with 50λ Ampicillin) and checked Optical Density. According to the O.D. value, we induced 37℃ and 30℃ sample and did sampling. Each samples were induced by different conditions ; 1mM and 2mM of IPTG, 10mM and 30mM of arabinose.

Back to the Calendar
September 9th 2012

 PACKMAN
Moth_0109 expression optimization _ gel picture
Results

0818Fig1
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 gel was ruined during staining/destaining procedure, and we couldn’t find any band for this protein.
Therefore, we concluded that expression of 0109 gene on pBAD vector has failed and decided to try once more.



Moth_1191 expression optimization _ gel picture
Results

0818Fig1


Discussion

For each samples of different induction conditions, we run SDS-PAGE gel and checked the presence of 33.92kDa protein, methylenetetrahydrofolate reductase. The gel was ruined during staining/destaining procedure, and we couldn’t find any band for this protein.
Therefore, we concluded that expression of 1191 gene on pBAD vector has failed and decided to try once more.



FDH pTrc expression optimization _ gel picture
Results

0818Fig1


Discussion

For each samples of different induction conditions, we run SDS-PAGE gel and checked the presence of 40.37kDa protein, Formate dehydrogenase. The gel was ruined during staining/destaining procedure, and we couldn’t find any band for this protein.
Therefore, we concluded that expression of FDH gene on pTrc vector has failed and decided to try once more.



FDH pBAD expression optimization _ gel picture
Results

0818Fig1


Discussion

For each samples of different induction conditions, we run SDS-PAGE gel and checked the presence of 40.37kDa protein, Formate dehydrogenase. The gel was ruined during staining/destaining procedure, and we couldn’t find any band for this protein.
Therefore, we concluded that expression of FDH gene on pTrc vector has failed and decided to try once more.



Primer design to PCR-amplify piBR181 vector.
Results

0818Fig1


Discussion

For the Gibson assembly with RE digestion of vector, we had several problems with de-phosphorylation and purification after de-phosphorylation. Therefore we decided to PCR-amplify the piBR181 vector and use the PCR product in Gibson assembly.

Back to the Calendar
September 10th 2012

 PACKMAN
PCR amplification of 1202 gene with new primers
Results

0818Fig1
Discussion

The correct size of band appeared for the second PCR product, but there was no band appeared for the first PCR product.

 Flip Flop

Bxb1_GTG_pBAD single cut check with different enzyme
Single cut with SnaBI

Results

2_0910Fig1

No desired size(5.6kb) vector appeared.
Back to the Calendar
September 11th 2012

 PACKMAN
PCR amplification of piBR181 vector
Results

0818Fig1
Discussion

The correct size of piBR181 vector pcr product is 5297bp. There appeared the right size and unspecific band on the gel. So we decided to do pcr once more.



Pre-culture of 0109, 1191, FDH containing cells.
Procedure

We did 5ml pre-culture on 10ml tube for the cells containing each gene. Cell was grown about 16hours after pre-culture, and then induced with proper inducer.

Back to the Calendar
September 12th 2012

 PACKMAN
PCR amplification of piBR181 vector : 10 sample
Results

0818Fig1
Discussion

The full PCR construct of piBR181 vector is 5297bp. From the gel image, we could find that right size of piBR181 PCR product clearly exists.



FDH pTrc & pBAD/0109 pBAD/1191 pBAD MG1655 expression check (sampling and induction)
Procedure

We seeded cells (500λ of cells put into 50mL LB with 50λ Ampicillin) and checked Optical Density. According to the O.D. value, we induced 37℃ and 30℃ sample and did sampling. Each samples were induced by different conditions ; 1mM and 2mM of IPTG, 10mM and 30mM of arabinose.

 Flip Flop

Bxb1_GTG_pBAD colony #4~7 inoculation, miniprep and single cut check Single cut with SnaBI
Single cut with SnaBI

Results

2_0912Fig1

Cloning completed
Back to the Calendar
September 13th 2012

 PACKMAN
Moth_0109 pBAD expression check
Results

0818Fig1
Discussion

For each samples of different induction conditions, we ran 10% SDS-PAGE gel and checked the presence of 59.94kDa protein, formate--tetrahydrofolate ligase. We couldn’t find any band for this protein.
There was no significant band on the gel, so we couldn’t conclude that this protein is expressed. Our opinion is that we should run Western blot to exactly find this protein.



Moth_1191 pBAD expression check
Results

0818Fig1


Discussion

For each samples of different induction conditions, we run 15% SDS-PAGE gel and checked the presence of 33.92kDa protein, methylenetetrahydrofolate reductase. Although we were not able to discriminate each band of insoluble fraction, We could find that there exists right band at the right size.



FDH pBAD expression check
Results

0818Fig1


Discussion

For each samples of different induction conditions, we run 8% SDS-PAGE gel and checked the presence of 40.37 kDa protein, Formate dehydrogenase. We couldn’t find any band for this protein.
There was no significant band on the gel, so we couldn’t conclude that this protein is expressed. Our opinion is that we should run Western blot to exactly find this protein.



FDH pTrc expression check
Results

0818Fig1


Discussion

For each samples of different induction conditions, we run 8% SDS-PAGE gel and checked the presence of 40.37 kDa protein, Formate dehydrogenase. We couldn’t find any band for this protein.
There was no significant band on the gel, so we couldn’t conclude that this protein is expressed. Our opinion is that we should run Western blot to exactly find this protein.

Back to the Calendar
September 14th 2012

 PACKMAN
Gibson assembly – assembly of pcr fragments (pcr fragment of vector added)
Results

Among three methods to assemble DNA fragments, we chose third method, which is assembly of PCR amplified DNA fragments and vector. 0.17umoles 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.

0818Fig1
0818Fig1

After 1 hour of incubation on 50℃, the vector was transformed into TOP10 and NEB competent cell. Cells were plated and grown overnight during colony formation.

Back to the Calendar
September 15th 2012

 Flip Flop

BBa_C0060, BBa_C0061, BBa_C0062 transformation into TOP10
Templates for Auto-regulated FlipFlop project.

Back to the Calendar
September 16th 2012

 PACKMAN
Gibson assembly colony pcr
Results

0818Fig1
Discussion

As we couldn’t get full fragment by colony-pcr, we did 3ml culture, plasmid mini-prep and PCR for each colony. This will take one more day to get result.

 Flip Flop

BBa_C0060, BBa_C0061, BBa_C0062, bFMO inoculation and enzyme cut check
BBa_C0060, BBa_C0061, BBa_C0062: from colony, EcoRI single cut

  • BBa_C0060: 2859bp
  • BBa_C0061: 2688bp
  • BBa_C0062: 2826bp
  • bFMO: from cell stock, EcoRI single cut


Results

2_0916#Fig#1

Back to the Calendar
September 17th 2012

 PACKMAN
Gibson assembly fragment PCR after prep
Results

0818Fig1
Discussion

There appeared correct size of band with several unspecific bands on the gel. We couldn’t even try to do gel extraction because the band was too thin and faint. We decided to do more rounds of pcr amplification.

 Flip Flop

Template preparation for OE PCR

Promoter construct: 210bp

Results

2_0917Fig1

Promoter PCR failed

2_0917Fig2
Back to the Calendar
September 18th 2012

 Flip Flop

Template preparation for OE PCR
Results

2_0918Fig1

Terminator PCR failed
Back to the Calendar
September 19th 2012

 PACKMAN
Gibson F1 PCR after prep
Results

0818Fig1
Discussion

We did PCR amplification after prep of F1 vector, but there appeared no band on the gel.



Gibson F2 5-1~5, F1-3-1~5, F1-4-1~5
Results

0818Fig1
Discussion

There appeared several unspecific bands on the gel and

 Flip Flop

Template preparation for OE PCR
Terminator: 179bp

Results

2_0919Fig1

→ Gel extracted
Back to the Calendar
September 20th 2012

 Flip Flop

Template preparation for OE PCR
Bxb1 Xis: 777bp

Results

2_0920Fig1

bFMO PCR w/ various condtions

2_0920Fig2



Bxb1_Xis-pGEM_B1 transfomation
Synthesized gene – Bxb1_Xis arrived and transformed into TOP10

Back to the Calendar
September 21st 2012

No Special Event!
Back to the Calendar
September 22nd 2012

 Flip Flop

Overlapping extension PCR
Results

2_0922Fig1

→ Gel extracted
Back to the Calendar
September 23rd 2012

 Flip Flop

Overlapping extension PCR-Nested PCR
Results

2_0923Fig1

Discussions

There was too little full construct nested. We think this is due to primer interfering by its LVA homology.


Overlapping extension PCR 2
Results

2_0923Fig2

Discussions

Bands were more intense than those of first trial.
Back to the Calendar
September 24th 2012

 Flip Flop

Overlapping extension PCR Gel Extraction
Results

2_0924Fig1



P1, PI construct
Results

Full fragment for pAuto is overlapped. The right size(3794bp) fragments are gel extracted.

0818Fig1
Back to the Calendar
September 25th 2012

No Special Event!
Back to the Calendar
September 26th 2012

 Flip Flop

Full pAuto and pAutoSimple Overlapping PCR
Results

0818Fig1


Discussion

The band intensity is too weak.


pAutoIntegrase Overlapping PCR
Results

0818Fig1

Full construct(2.5kb) for pAutoIntegrase is overlapped. The products are pcr purified and performed nested-pcr. 4th lane is the OE pcr product with rearranged template concentration.

0818Fig1


Template preparation of terminator
Results

0818Fig1

Terminator template for OE pcr is re-amplified with pfu-X DNA polymerase. The products are pcr purified.
Back to the Calendar
September 27th 2012

 PACKMAN
Pre-culture of Gibson F1, F2 colonies
Results

We checked that there were several colonies on the plate. 10 colonies for each fragment were picked, and culture into 3mL LB media containing 30ul Kanamycin.

Back to the Calendar
September 28th 2012

 PACKMAN
Vector preparation and PCR
Results

For each cultured cells, vectors were mini-prepped, and pcr was done with proper primers (F1 : 1201 F, 1197R, F2 : 1198F, 1516R)

Back to the Calendar
September 29th 2012

 Flip Flop

Template preparation of bFMO (for pAuto and pAutoSimple)
Results

0818Fig1

To find out the right temperature for primer binding, we performed gradient pcr from 45℃~55℃. But, all the pcr products showed non-specific binding of primers, and we selected the most proper temperature.


Overlapping extension PCR Gel Extraction
Results

0818Fig1


Back to the Calendar
September 30th 2012

 Flip Flop

Template vector preparation – double digestion check with EcoRI and PstI
Results

0818Fig1

Vector(BBa_J04450-pSB4A5) which will be used for cloning of pAutoIntegrase.


bFMO template amplification with optimized condition
Results

0818Fig1


pAutoIntegrase cloning check
Results

PCR amplified from prepped vector
0818Fig1
Back to the Calendar


Kaist Footer