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


Memo-Cell project

Recombination assay of HK022 and Lambda mutated recombination-sites.

In our project, we mutated the recombination sites of Phage Lambda and Phage HK022.
We wished to assess their recombination efficiency compared to the wild-type sites.
We also mutated the arms of the transposon Tn916, and performed experiments to assess its excision efficiency compared to the wild-type transposon.

To measure the recombination efficiency of our mutated phage recombination sites, we cloned these sites within a pir dependent plasmid (R6K ori) that could only be replicated in pir+ strains. These plasmids also carried the Chloramphenicol and Kanamycin resistance cassettes.

Integration assessment

We transformed a pir- strain expressing the Integrase under the control of a pBAD promoter (using our biobricks K329028 pBAD_IntHK, K329027 pBAD_IntLambda) with the corresponding plasmid carrying either the wild-type or the mutated attP site, and plated the bacteria on LB plates with and without the selective antibiotics.

Our experiment showed no real defect in the integration of the plasmids carrying the mutated attP sites for both HK022 and Lambda systems!

Excision assessment

We took the clones that had integrated the plasmids carrying the resistance cassettes, and checked the recombination sites that had been reformed following the integration (attL and attR) by sequencing.
We then transformed these positive clones with a plasmid carrying the corresponding phages enzymes that mediate excision of the phage from the bacterial chromosome: Xis and Int HK022 or Lambda.

We then plated the transformants on LB without any selection marker. We replicated the plates on a new plate with no antibiotics and with antibiotics (CmR and Kana). Bacteria that have excised the plasmid should not be able to grow on the medium with antibiotics.
This results here showed that mutated phage recombination site also efficiently mediated excision, although not quite at wild-type ones. This might be due to the mutation in the Xis binding sites, which have been showed to affect the excision efficiency.

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 overnight 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 Kanamycin. Colonies which have excised will grow on glucose but not on Kanamycin. 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 sites 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.