Team:KIT-Kyoto/Notebook-week7

From 2012.igem.org

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By red eye screening, we identified the strain 13 and strain 14 to be the successfully transformed lines. Photographs for the transformed red (orange) eye fly (strain 13) were taken.
By red eye screening, we identified the strain 13 and strain 14 to be the successfully transformed lines. Photographs for the transformed red (orange) eye fly (strain 13) were taken.
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Drosophila S2 cells (2.5 X 10<sup>5</sup> cells per well) were plated on the 24 well plate and cultured in the Schneider’s Drosophila medium containing 10% fetal bovine serum at 25 ℃.
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<h2>September 20th</h2>
<h2>September 20th</h2>
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By red eye screening, we identified the strain 23 and strain 29 to be the successfully transformed lines.
By red eye screening, we identified the strain 23 and strain 29 to be the successfully transformed lines.
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At 20 min after removing the medium, 25 uL of serum free medium containing the 1 uL of siLentfect and 200 ng each of pAct5C-GAL4 DNA and pUAS-EGFP DNA was added to the well. As a negative control, 25 uL of serum free medium containing the 1 uL of siLentfect and 200 ng of pUAS-EGFP DNA alone was added to the well. Similarly, 25 uL of serum free medium containing the 1 uL of siLentfect and 200 ng each of pAct5C-GAL4 DNA and pUAS-LacZ DNA was added to the well. As a negative control for this experiment, 25 uL of serum free medium containing the 1 uL of siLentfect and 200 ng of pUAS-LacZ DNA alone was added to the other well. At 4 hours after transfection, the transfection medium was removed and the Schneider’s Drosophila medium containing 10% fetal bovine serum was added to each well. The cells were incubated for 48 or 72 hours at 25 ℃.
 
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<h2>September 22nd</h2>
<h2>September 22nd</h2>
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Red eye screening was continued.
Red eye screening was continued.
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The cells transfected with pAct5C-GAL4 DNA and pUAS-EGFP DNA were examined under the confocal lazer microscope (Olympus, Fv10i).
 
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Results: 12.6% cells were observed to be EGFP-positive, while only o.5% cells were apparently EGFP-positive for the negative control cells, indicating that the P-element plasmid, pUAS-EGFP DNA, Biobrick part (BBa_K758006) is truly functional in Drosophila cells. We therefore decided to send this prasmid to iGEM Headquarters.
 
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Leftpanel: cells co-transfected with pAct5C-GAL4 DNA and pUAS-EGFP DNA
 
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Right panel: cells transfected with pUAS-EGFP DNA alone (negative control)
 
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    <img src="http://2012.igem.org/wiki/images/b/b5/実験ノート1.png" width="282" height="282">  <img src="http://2012.igem.org/wiki/images/c/cd/実験ノート2.png" width="282" height="282">
 
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The cells transfected with pAct5C-GAL4 DNA and pUAS-LacZ DNA were fixed with cold methanol for 20 min or 4% paraformaldehyde for 15 min. After fixation, cells were washed with 0.5% Triton X-100 in PBS, then masked with 5% normal goat serum in PBS for 1 hour. Then the monoclonal anti-beta galactosidase antibody (X500 dilution in PBS containing 5% normal goat serum) was added to the fixed cells and incubated for 16 hours at 4℃.
 
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<h2>September 23rd</h2>
<h2>September 23rd</h2>
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By red eye screening, we identified the strain 56 and strain 65 to be the successfully transformed lines. These transformed flies are kept at 25℃ and later will be further inspected for chromosome linkage of the transgene.  
By red eye screening, we identified the strain 56 and strain 65 to be the successfully transformed lines. These transformed flies are kept at 25℃ and later will be further inspected for chromosome linkage of the transgene.  
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After incubation with the first antibody, the cells were washed with PBS (three times for 10 min each). Then the Alexa-conjugated anti-mouse IgG (X400 dilution in PBS containing 5% normal goat serum) added to the cells and incubate for 4 hours at 4℃. After washing with PBS (three times for 10 min each), cells were mounted with mounting medium in DAPI and examined under the confocal lazer microscope (Olympus, Fv10i).
 
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Results: 10.1% cells co-transfected with pAct5C-GAL4 DNA and pUAS-LacZ DNA were observed to be LacZ-positive, while no LacZ-positive cell was detected for the negative control cells, indicating that the P-element plasmid, pUAS-LacZ DNA, Biobrick part (BBa_K758005) is truly functional in Drosophila cells. We therefore decided to send this plasmid to iGEM Headquarters.
 
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After 72 hours incubation, the cells transfected with pAct5C-GAL4 DNA and pUAS-EGFP DNA were examined under the confocal lazer microscope (Olympus, Fv10i). Results were nearly the same as those after 48 hours incubation.
 
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Leftpanel: cells co-transfected with pAct5C-GAL4 DNA and pUAS-LacZ DNA
 
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Right panel: cells transfected with pUAS-LacZ DNA (negative control)
 
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    <img src="http://2012.igem.org/wiki/images/a/a8/実験ノート3.png" width="282" height="282">  <img src="http://2012.igem.org/wiki/images/1/17/実験ノート4.png" width="282" height="282">
 
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Revision as of 11:48, 25 September 2012






September 18th


By red eye screening, we identified the strain 7 and strain 10 to be the successfully transformed lines. Photographs for the transformed red (yellow) eye fly (strain 10) were taken.

September 19th


By red eye screening, we identified the strain 13 and strain 14 to be the successfully transformed lines. Photographs for the transformed red (orange) eye fly (strain 13) were taken.

September 20th


By red eye screening, we identified the strain 23 and strain 29 to be the successfully transformed lines.

September 21st


Red eye screening was continued.

September 22nd


Red eye screening was continued.

September 23rd


By red eye screening, we identified the strain 56 and strain 65 to be the successfully transformed lines. These transformed flies are kept at 25℃ and later will be further inspected for chromosome linkage of the transgene.

September 24th




September 25th




September 26th