Team:KAIST Korea/Notebook Labnote/2012 10

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<span id="little">Each vector was prepped and cut with HindIII, NotI to see if proper assembly product has formed. </span></br>
<span id="little">Each vector was prepped and cut with HindIII, NotI to see if proper assembly product has formed. </span></br>
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<span id='little'>Table below is composition of each enzyme reaction.</span>
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<span id='little'>Table below is composition of each enzyme reaction.</span></br>

Latest revision as of 03:01, 27 October 2012

KAIST Korea 2012 iGEM

Notebook : Labnote-October

Labnote

October

October 1st 2012

 PACKMAN

Plasmid gel electrophoresis
Results

1_1001Fig1

Plasmid of Gibson fragment 1 (sample 1~10) were run on the gel to see if there is any problem with plasmid mini-prep process.

Discussions

It seems that this vector is supercoiled form, of which the size is about 10000bp.
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October 2nd 2012

 Flip Flop

bFMO template preparation with new primer
Results

0818Fig1

We have found that our bFMO sequence was wrong. So that we have ordered new primers with right sequence.


OE PCR template preparation
Results

0818Fig1


pAutoIntegrase cloning check
Results

0818Fig1


Discussion

We have checked the result of cloning with various ways but we can’t sure about the success of cloning.

 PACKMAN

Restriction enzyme cut
Results

Fragment 1 was digested with Hind III and Not I. Digestion of vector generates four fragments, the size of each fragments are as follows: 15280bp, 6593bp, 5748bp, 3000bp. Fragment 2 was digested with Not I and Xho I. Digestion of vector generates three fragments. The size of each fragments are as follows : 11213bp, 9010bp, 2207bp.
Table below is composition of each enzyme reaction.

1_1002Fig1

1_1002Fig2

Discussions

Different from our prediction, bands appeared at the wrong size, about the exact size of the vector. Therefore, we could conclude that colonies F1 1~10 and F2 1~5 contain background vectors.


Restriction enzyme cut _ gel 2
Results

1_1002Fig3

Discussions

For colony 6, 8, and 10, there appeared correct size of whole vector size. It seemed that Not I or Xho I was not active on the reaction condition and only one of those two enzymes cut circular DNA into linear DNA.


Pre-culture of Gibson F1 colonies
Additional 10 colonies for fragment 1 were picked, and culture into 3mL LB media containing 30ul Kanamycin.
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October 3rd 2012

 Flip Flop

Visualization of inversion from GFP to RFP
Procedure


bfmo gene is constructed in the vector(pJ401, Km registance) and transformed into DH5α.
50λ cell from stock is inoculated in 5mL LB


Visualization of inversion from GFP to RFP
Procedure
We sampled the cell every 3hr. We induced one group of samples and un-induced another group of samples as a control. We took the picture of cell with confocal microscope at absorbance 500nm for GFP expression and 600nm for RFP expression.

Results

0818Fig1


Discussion

We obtained proper data. Un-induced sample doesn’t show measurable RFP expression throughout the culture. However, induced sample shows the increase of RFP expression. Increase of GFP level in induced sample is due to the stability of GFP and time delay before inversion.


Insert DNA preparation with OE pcr
Results

0818Fig1


Cloning
Results

0818Fig1
0818Fig1
 PACKMAN

Vector preparation and Enzyme cut
For each cultured cells, vectors were mini-prepped, and we did enzyme cut with those vectors.
Table below is composition of each enzyme reaction.

Results

1_1003Fig1


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October 4th 2012

 PACKMAN

Enzyme cut result
Results

1_1004Fig1

It seems there are only background vectors in the cells.
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October 5th 2012

No Special Event!
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October 6th 2012

No Special Event!
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October 7th 2012

No Special Event!
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October 8th 2012

 PACKMAN

Pre-culture of Gibson assembly fragment1 containing colony
Results

Colonies number 16 through 25 were cultured in 3mL LB containing 1000x Kanamycin (30ul). Cells were cultured about 16 hours and vector was mini-prepped.
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October 9th 2012

 PACKMAN

Vector mini-prep and enzyme cut of prepped vector.
Results

Each vector was prepped and cut with HindIII, NotI to see if proper assembly product has formed.
Table below is composition of each enzyme reaction.

1_1009Fig1

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October 10th 2012


 Flip Flop

LuxI and Xis OE template preparation
Results

2_1010Fig1



pAutoIntegrase cloning check and Xis template vector prep
Results

2_1010Fig2

pAutoIntegrase 1-10 – HindIII cut
pGEM-Xis 1-2 – EcoRI cut



insert DNA preparation with OE PCR
Results

2_1010Fig3

┗ pAutoSimple(1-3) and pAutoSimpleInverted(4-6)

2_1010Fig4

┗ pAutoSimple and pAutoSimpleInverted (1 and 2, respectively)/ pAuto(1-3) and pAutoInverted(4-6)
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October 11th 2012


 Flip Flop

Additional templates
Results

2_1011Fig1

┗ Integrase and Excisionase template

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October 12th 2012

No Special Event!
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October 13th 2012

No Special Event!
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October 14th 2012


 Flip Flop

pAutoIntegrase Cloning
Insert DNA – OE PCR

Results

2_1014Fig1

Cloning into pSB4A5 is performed with gel extracted DNA fragment.

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October 15th 2012

No Special Event!
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October 16th 2012


 Flip Flop

pAutoSimple inserts nested PCR and aiiA template
Results

2_1016Fig1



pAuto, pAutoInverted, pAutoSimple, pAutoSimpleInverted miniprep and RE cut
Results

2_1016Fig2

single digestion with HindIII.
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October 17th 2012


 Flip Flop

pAuto OE PCR and Xis, simple bFMO, and bFMO template preparation
Results

2_1017Fig1

sizes ok


pAuto insert DNA preparation
Results

2_1017Fig2

intermediate fragment nested PCR

2_1017Fig3

Full pAuto insert DNA construct OE PCR

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October 18th 2012


 Flip Flop

pAutoIntegrase enz cut check with NotI
Results

2_1018Fig1

Lane 3, 6, 8 seem to be cloned.


Cell culture in 96-well plate for Indigo expression time optimization
Procedure


We set the O.D value to 0.1 and cultured 1.5ml cell in 96 deep well plate. At each time point, we transferred 1ml cell from 96 deep well plate to EP tube and 200λ cell to flat 96 well plate for O.D check.
After cell down in EP tube, we discarded the supernatant and resuspended the pellet in 200λ DMSO. We did sonication to the sample and after another cell down, we took the supernatant for TECAN measurement. Also, we checked absorbance after 3 days incubation and before 3days incubation.
We used TECAN at absorbance 620nm to measure the quantity of indigo production

Results

0818Fig1



We subtracted absorbance value of pure LB from value of samples and we set all samples to have same amount of cell using O.D value
Graph shown below is indigo intensity per cell. After 3 days incubation, indigo seems to be degraded and all the samples seem to have almost same indigo level.

0818Fig1


We excluded 0hr data from the graph to see teh rising tendency clearly.


Discussion


This data shows that the cell successfully produces indigo. We can use this gene as a reporter of our module. However, we only checked the increase of indigo. It may decrease after prolonged culture. We should do additional experiments to get the peak in the graph and optimize the production time of indigo.
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October 19th 2012


 Flip Flop

pAutoIntegrase Alternative sequencing with fragments amplification
Results

2_1019Fig1

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October 20th 2012

No Special Event!
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October 21st 2012

No Special Event!
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October 22nd 2012

No Special Event!
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October 23rd 2012

No Special Event!
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October 24th 2012


 Flip Flop

OE template preparation
Results

2_1024Fig1

gp47, aiiA, LuxI, bFMO for simple and pAuto

2_1024Fig1

promoter and Inverted one

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October 25th 2012

 Flip Flop

pAuto intermediate fragment OE PCR
Results

1025Fig1


OE PCR with new primer
Results

2_1025Fig2

pAutoSimple/inverted

2_1025Fig2

pAuto/inverted

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October 26th 2012

No Special Event!
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October 27th 2012

No Special Event!
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October 28th 2012

No Special Event!
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October 29th 2012

No Special Event!
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October 30th 2012

No Special Event!
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October 31st 2012

No Special Event!
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