Memo-Cell project

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Recombination assay of HK022 and Lambda mutated recombination-sites.

Test of the excision by Tn916

Errors bars indicate the standard deviation from two independent trials.
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).

To check the excision, we have done colony PCR for several clones of each transposon version with primers matching in both site of the attB site.
We can see that for this clones, there is a band around 2800 bp which correspond to the plasmid integrated (5000 bp) without the transposon (2200 bp).
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.