|
|
| Line 2: |
Line 2: |
| | <partinfo>BBa_K329055 short</partinfo> | | <partinfo>BBa_K329055 short</partinfo> |
| | | | |
| − | ==Test of the excision by Tn916==
| |
| − |
| |
| − | <html><p style="display:block;">
| |
| − |
| |
| − | <img src="https://static.igem.org/mediawiki/2010/5/5e/Transposase_assay_protocol-01.jpg" width=40%>
| |
| − |
| |
| − | <center>
| |
| − | <br>
| |
| − | <img src="https://static.igem.org/mediawiki/2010/9/92/Excision.PNG" width=70%>
| |
| − | </center>
| |
| − | <br>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.
| |
| − | <br>
| |
| − |
| |
| − | 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).
| |
| − |
| |
| − | <br>
| |
| − | <br>
| |
| − |
| |
| − | 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.
| |
| − | <br>
| |
| − | 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).
| |
| − |
| |
| − | <center>
| |
| − | <br>
| |
| − | <img src="https://static.igem.org/mediawiki/2010/b/b9/Gel.PNG" width=70%>
| |
| − | </center>
| |
| − |
| |
| − |
| |
| − | 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.
| |
| − |
| |
| − |
| |
| − | </br>
| |
| − |
| |
| − | </center>
| |
| − | <br /><br />
| |
| − | </p>
| |
| − |
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | </html>
| |
| | <!-- Add more about the biology of this part here | | <!-- Add more about the biology of this part here |
| | ===Usage and Biology=== | | ===Usage and Biology=== |
