Team:Paris Liliane Bettencourt/Project/Memo-cell/Results

From 2010.igem.org

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<img src="https://static.igem.org/mediawiki/2010/9/92/Excision.PNG" width=70%>
 
<img src="https://static.igem.org/mediawiki/2010/5/5c/Integration_results-01.jpg" width=70%>
<img src="https://static.igem.org/mediawiki/2010/5/5c/Integration_results-01.jpg" width=70%>
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<img src="https://static.igem.org/mediawiki/2010/9/92/Excision.PNG" width=70%>
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To determine the efficiency of excission with the different arms we decided to
 +
 +
use a Kan Lacz counter selection system. We took culture of cells where one of
 +
 +
the third mutated transposon had been integrated via recombination of attP and
 +
 +
attB site (integrase Lambda). This cells have been transform with a plasmid
 +
 +
carrying the both gene Int and Xis of the Tn916 under the control of inducible
 +
 +
Pbad promoter. From an overnigth culture, dilution has been done and induction at
 +
 +
the beginning of exponential phase (O.D 0.2) with Arabinose at final
 +
 +
concentration of 0.2%. Every two hours, we took one sample of each culture and
 +
 +
put them in 2% of glucose to inhibit the Pbad promoter. Then during one hour the
 +
 +
sample is kept at 37°C to dilute protein of the transposase. Like this we
 +
 +
decrease the probability to have event of excision between the sampling and
 +
 +
plating. To permit counting of colonies several dilution where plated in glucose
 +
 +
1% and then replicate on plate with Kanamycine. Colonies which have excise will
 +
 +
not grow in Kanamycine. The excision efficiency correspond to 1-(unexcised
 +
 +
CFU/Total CFU) number of cells Kanamycine resistante (not excissed) over total
 +
 +
number of cells).
 +
 +
 +
What we observe is that the Wild type and Lambda arms of the Tn916 permit an
 +
 +
excision of 100% after 2h of induction whereas the HK arms have an efficiency of
 +
 +
55% after 30h of induction. The results for wild type are consistent with the
 +
 +
bibliography exept that the maximum efficiency is rise faster in our system.
 +
Errrors bars indicate the standard deviation from two independent trials.
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Revision as of 23:52, 27 October 2010


Memo-Cell project

To determine the efficiency of excission with the different arms we decided to use a Kan Lacz counter selection system. We took culture of cells where one of the third mutated transposon had been integrated via recombination of attP and attB site (integrase Lambda). This cells have been transform with a plasmid carrying the both gene Int and Xis of the Tn916 under the control of inducible Pbad promoter. From an overnigth culture, dilution has been done and induction at the beginning of exponential phase (O.D 0.2) with Arabinose at final concentration of 0.2%. Every two hours, we took one sample of each culture and put them in 2% of glucose to inhibit the Pbad promoter. Then during one hour the sample is kept at 37°C to dilute protein of the transposase. Like this we decrease the probability to have event of excision between the sampling and plating. To permit counting of colonies several dilution where plated in glucose 1% and then replicate on plate with Kanamycine. Colonies which have excise will not grow in Kanamycine. The excision efficiency correspond to 1-(unexcised CFU/Total CFU) number of cells Kanamycine resistante (not excissed) over total number of cells). What we observe is that the Wild type and Lambda arms of the Tn916 permit an excision of 100% after 2h of induction whereas the HK arms have an efficiency of 55% after 30h of induction. The results for wild type are consistent with the bibliography exept that the maximum efficiency is rise faster in our system. Errrors bars indicate the standard deviation from two independent trials.


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