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Team:Paris Liliane Bettencourt/Project/Memo-cell/Results
From 2010.igem.org
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.
