"
Team:KAIST Korea/Notebook Labnote/2012 10
From 2012.igem.org
2012 KAIST Korea
Mail : kaist.igem.2012@gmail.com
Twitter : twitter.com/KAIST_iGEM_2012
Facebook : www.facebook.com/KAISTiGEM2012
Notebook : Labnote-October
Labnote
OctoberOctober 1st 2012
No Special Event!
Back to the Calendar
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.
Flip Flop
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.
Back to the Calendar
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
Back to the Calendar
October 4th 2012
No Special Event!
Back to the Calendar
October 5th 2012
No Special Event!
Back to the Calendar
October 6th 2012
No Special Event!
Back to the Calendar
October 7th 2012
No Special Event!
Back to the Calendar
October 8th 2012
No Special Event!
Back to the Calendar
October 9th 2012
No Special Event!
Back to the Calendar
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)
Back to the Calendar
October 11th 2012
Flip FlopAdditional templates
Results
Back to the Calendar
┗ Integrase and Excisionase template
October 12th 2012
No Special Event!
Back to the Calendar
October 13th 2012
No Special Event!
Back to the Calendar
October 14th 2012
Flip FloppAutoIntegrase Cloning
Insert DNA – OE PCR
Results
Back to the Calendar
Cloning into pSB4A5 is performed with gel extracted DNA fragment.
October 15th 2012
No Special Event!
Back to the Calendar
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.
Back to the Calendar
October 17th 2012
Flip FloppAuto OE PCR and Xis, simple bFMO, and bFMO template preparation
Results
sizes ok
pAuto insert DNA preparation
Results
Back to the Calendar
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.
Back to the Calendar
October 19th 2012
Flip FloppAutoIntegrase Alternative sequencing with fragments amplification
Results
Back to the Calendar
October 20th 2012
No Special Event!
Back to the Calendar
October 21st 2012
No Special Event!
Back to the Calendar
October 22nd 2012
No Special Event!
Back to the Calendar
October 23rd 2012
No Special Event!
Back to the Calendar
October 24th 2012
Flip FlopOE template preparation
Results
Back to the Calendar
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
Back to the Calendar
pAutoSimple/inverted
pAuto/inverted
October 26th 2012
No Special Event!
Back to the Calendar
October 27th 2012
No Special Event!
Back to the Calendar
October 28th 2012
No Special Event!
Back to the Calendar
October 29th 2012
No Special Event!
Back to the Calendar
October 30th 2012
No Special Event!
Back to the Calendar
October 31st 2012
No Special Event!
Back to the Calendar
