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

From 2010.igem.org

(Difference between revisions)

Revision as of 00:53, 28 October 2010

Memo-Cell project

Test of HK and Lambda Integrase

Test of the excision by Tn916

To determine the efficiency of excision with the different arms we decided to use a Kan LacZ counter selection system (see design section : mutated Tn916). We took a culture of cells where one of the third transposon (Wild Type, HK, Lambda) had been integrated via recombination of attP and attB site (integrase Lambda). This cells have been transformed with a plasmid carrying both the Tn916 Int and Xis genes. These genes are under the control of the inducible Pbad promoter. We then diluted an overnigth culture and subsequently induced at the beginning of exponential phase (O.D 0.2) with Arabinose at final concentration of 0.2%. Following the induction, we took one sample of each culture every 2 hours and put them in 2% of glucose to inhibit the Pbad promoter for 1 hour at 37°C. Hence, we acheived the dilution of the transposase enzyme. The aim of this experimental step is to decrease the probability to have an event of excision between sampling and plating.
To permit counting of colonies several dilutions where plated on glucose 1% and then replicated on a plate with Kanamycine. Colonies which have excised will grow on glucose but not on Kanamycine. The excision efficiency correspond to 1-(unexcised CFU/Total CFU). 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 a much lower efficiency of 55% after 30h of induction. The results for wild type are consistent with the bibliography except that the maximum efficiency is reached faster in our system. This is probably due to the fact the transpose is put on a high copy plasmid in trans. Errors bars indicate the standard deviation from two independent trials.

@@ Line 61: / Line 61: @@
 <img src="https://static.igem.org/mediawiki/2010/9/92/Excision.PNG" width=70%>
 </center>
-<br>To determine the efficiency of excission with the different arms we decided to
+<br>To determine the efficiency of excision with the different arms we decided to
-use a Kan Lacz counter selection system. We took culture of cells where one of
+use a Kan LacZ counter selection system (see design section : mutated Tn916). We took a culture of cells where one of
-the third mutated transposon had been integrated via recombination of attP and
+the third transposon (Wild Type, HK, Lambda) had been integrated via recombination of attP and
-attB site (integrase Lambda). This cells have been transform with a plasmid
+attB site (integrase Lambda). This cells have been transformed with a plasmid
-carrying the both gene Int and Xis of the Tn916 under the control of inducible
+carrying both the Tn916 Int and Xis genes. These genes are under the control of the inducible
-Pbad promoter. From an overnigth culture, dilution has been done and induction at
+Pbad promoter. We then diluted an overnigth culture and subsequently induced 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
+concentration of 0.2%. Following the induction, we took one sample of each culture every 2 hours and
-put them in 2% of glucose to inhibit the Pbad promoter. Then during one hour the
+put them in 2% of glucose to inhibit the Pbad promoter for 1 hour at 37°C. Hence, we acheived the dilution of the transposase enzyme. The aim of this experimental step is to decrease the probability to have an event of excision between sampling and plating.
+<br>
-sample is kept at 37°C to dilute protein of the transposase. Like this we
+To permit counting of colonies several dilutions where plated on glucose
-decrease the probability to have event of excision between the sampling and
+% and then replicated on a plate with Kanamycine. Colonies which have excised will
-plating. To permit counting of colonies several dilution where plated in glucose
+grow on glucose but not on Kanamycine. The excision efficiency correspond to 1-(unexcised
-% and then replicate on plate with Kanamycine. Colonies which have excise will
+CFU/Total CFU).
-not grow in Kanamycine. The excision efficiency correspond to 1-(unexcised
+What we observe is that the Wild type and Lambda arms of the Tn916 permit an
-CFU/Total CFU) number of cells Kanamycine resistante (not excissed) over total
+excision of 100% after 2h of induction whereas the HK arms have a much lower efficiency of
-number of cells).
+% after 30h of induction. The results for wild type are consistent with the
+bibliography except that the maximum efficiency is reached faster in our system. This is probably due to the fact the transpose is put on a high copy plasmid in trans.
-What we observe is that the Wild type and Lambda arms of the Tn916 permit an
+Errors bars indicate the standard deviation from two independent trials.
-excision of 100% after 2h of induction whereas the HK arms have an efficiency of
-% 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.
 </br>