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Team:KAIST Korea/Notebook Labnote/2012 10
From 2012.igem.org
2012 KAIST Korea
Mail : kaist.igem.2012@gmail.com
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Notebook : Labnote-October
Labnote
OctoberOctober 1st 2012
PACKMANPlasmid gel electrophoresis
Results
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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Back to the CalendarOctober 2nd 2012
Flip FlopbFMO template preparation with new primer
Results
We have found that our bFMO sequence was wrong. So that we have ordered new primers with right sequence.
PACKMAN
Flip Flop
PACKMAN
PACKMAN
PACKMAN
PACKMAN
Flip Flop
Flip Flop
Flip Flop
Flip Flop
Flip Flop
Flip Flop
Flip Flop
Flip Flop
Flip Flop
OE PCR template preparation
Results
pAutoIntegrase cloning check
Results
Discussion
We have checked the result of cloning with various ways but we can’t sure about the success of cloning.
PACKMANRestriction 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.
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
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 FlopVisualization 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
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
Cloning
Results
PACKMANVector 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
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October 4th 2012
PACKMANEnzyme cut result
Results
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
PACKMANPre-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
PACKMANVector 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.
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October 10th 2012
Flip FlopLuxI and Xis OE template preparation
Results
pAutoIntegrase cloning check and Xis template vector prep
Results
pAutoIntegrase 1-10 – HindIII cut
pGEM-Xis 1-2 – EcoRI cut
insert DNA preparation with OE PCR
Results
┗ pAutoSimple(1-3) and pAutoSimpleInverted(4-6)
┗ pAutoSimple and pAutoSimpleInverted (1 and 2, respectively)/ pAuto(1-3) and pAutoInverted(4-6)
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October 11th 2012
Flip FlopAdditional templates
Results
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┗ Integrase and Excisionase template
October 12th 2012
No Special Event!
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October 13th 2012
No Special Event!
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October 14th 2012
Flip FloppAutoIntegrase Cloning
Insert DNA – OE PCR
Results
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Cloning into pSB4A5 is performed with gel extracted DNA fragment.
October 15th 2012
No Special Event!
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October 16th 2012
Flip FloppAutoSimple inserts nested PCR and aiiA template
Results
pAuto, pAutoInverted, pAutoSimple, pAutoSimpleInverted miniprep and RE cut
Results
single digestion with HindIII.
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October 17th 2012
Flip FloppAuto OE PCR and Xis, simple bFMO, and bFMO template preparation
Results
sizes ok
pAuto insert DNA preparation
Results
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intermediate fragment nested PCR
Full pAuto insert DNA construct OE PCR
October 18th 2012
Flip FloppAutoIntegrase enz cut check with NotI
Results
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
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.
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 FloppAutoIntegrase Alternative sequencing with fragments amplification
Results
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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 FlopOE template preparation
Results
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gp47, aiiA, LuxI, bFMO for simple and pAuto
promoter and Inverted one
October 25th 2012
Flip FloppAuto intermediate fragment OE PCR
Results
OE PCR with new primer
Results
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pAutoSimple/inverted
pAuto/inverted
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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