Difference between revisions of "Part:BBa K329035"

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==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>
 
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<img src="https://static.igem.org/mediawiki/2010/9/92/Excision.PNG" width=70%>
 
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<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>
 
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<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.
 
 
 
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Revision as of 16:38, 30 October 2010

pBAD/araC - RBS - Xis_Int Tn916

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1205
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1144
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 979
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 961