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| − | ==Test of the excision by Tn916==
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| − | <html><p style="display:block;">
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| − | <img src="https://static.igem.org/mediawiki/2010/5/5e/Transposase_assay_protocol-01.jpg" width=40%>
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| − | <center>
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| − | <br>
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| − | <img src="https://static.igem.org/mediawiki/2010/9/92/Excision.PNG" width=70%>
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| − | </center>
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| − | <br>To determine the efficiency of excision with the different arms we decided to
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| − | use a Kan LacZ counter selection system (see design section : mutated Tn916). We took a culture of cells where one of
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| − | the third transposon (Wild Type, HK, Lambda) had been integrated via recombination of attP and
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| − | attB site (integrase Lambda). This cells have been transformed with a plasmid
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| − | carrying both the Tn916 Int and Xis genes. These genes are under the control of the inducible
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| − | Pbad promoter. We then diluted an overnigth culture and subsequently induced at
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| − | the beginning of exponential phase (O.D 0.2) with Arabinose at final
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| − | concentration of 0.2%. Following the induction, we took one sample of each culture every 2 hours and
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| − | 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.
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| − | <br>
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| − | To permit counting of colonies several dilutions where plated on glucose
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| − | 1% and then replicated on a plate with Kanamycine. Colonies which have excised will
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| − | grow on glucose but not on Kanamycine. The excision efficiency correspond to 1-(unexcised
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| − | CFU/Total CFU).
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| − | <br>
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| − | <br>
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| − | 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.
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| − | <br>
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| − | 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).
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| − | <center>
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| − | <br>
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| − | <img src="https://static.igem.org/mediawiki/2010/b/b9/Gel.PNG" width=70%>
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| − | </center>
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| − | What we observe is that the Wild type and Lambda arms of the Tn916 permit an
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| − | excision of 100% after 2h of induction whereas the HK arms have a much lower efficiency of
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| − | 55% after 30h of induction. The results for wild type are consistent with the
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| − | 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.
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| − | Errors bars indicate the standard deviation from two independent trials.
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| − | </br>
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| − | </center>
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| − | <br /><br />
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| − | </p>
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| − | </div>
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| − | </div>
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| − | </html>
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| | <!-- Add more about the biology of this part here | | <!-- Add more about the biology of this part here |
