Difference between revisions of "Part:BBa K329055"

Latest revision as of 19:23, 5 November 2010

[KanR + LacZ] in between MUT_MUT[Lambda] Tn916 Arms

This part is composed of a Kanamycine resistance and LacZ cassette between two mutated arms (left and right) of the transposon Tn916 containing each of us one half of the attR lambda recombination site. When the both enzyme of the transposon,Int and Xis tn916 are expressed (see BBa_K329035 and BBa_K329018)., the both arms link together forming a circular DNA sequence excised from the hosting DNA (plasmid or chromosome). The excision permit to create a functionnal attR lambda recombination site. This mutant provide a high efficiency excision characterized (see experience part).

Usage and Biology

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal SpeI site found at 363
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 7
Illegal NheI site found at 30
Illegal NheI site found at 382
Illegal NheI site found at 405
Illegal SpeI site found at 363
21
COMPATIBLE WITH RFC[21]
23
INCOMPATIBLE WITH RFC[23]
Illegal SpeI site found at 363
25
INCOMPATIBLE WITH RFC[25]
Illegal SpeI site found at 363
1000
COMPATIBLE WITH RFC[1000]

@@ Line 2: / Line 2: @@
 <partinfo>BBa_K329055 short</partinfo>
-==Test of the excision by Tn916==
+This part is composed of a Kanamycine resistance and LacZ cassette between two mutated arms (left and right) of the transposon Tn916 containing each of us one half of the attR lambda recombination site.
+When the both enzyme of the transposon,Int and Xis tn916 are expressed (see <partinfo>BBa_K329035</partinfo> and <partinfo>BBa_K329018</partinfo>)., the both arms link together forming a circular DNA sequence excised from the hosting DNA (plasmid or chromosome). The excision permit to create a functionnal attR lambda recombination site.
+This mutant provide a high efficiency excision characterized (see experience part).
-<html><p style="display:block;">
+===Usage and Biology===
-<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
-% 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
-% 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
-===Usage and Biology===
 <!-- -->
 <span class='h3bb'>Sequence and Features</span>
 <partinfo>BBa_K329055 SequenceAndFeatures</partinfo>
 <!-- Uncomment this to enable Functional Parameter display