Difference between revisions of "Part:BBa K1132038:Experience"

 
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This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
 
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how you used this part and how it worked out.
 
  
 
===Applications of BBa_K1132038===
 
===Applications of BBa_K1132038===
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===In vitro characterization of BBa_K1132036===
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The basic idea is to perform the switch with a cell lysis containing the recombinase and the plasmid containing the gate to be switched. After incubation, transformation of the plasmid containing the gate allows the quantification of switched versus non switched plasmids. Experiments have been performed as described in the protocol.
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[[http://2013.igem.org/Team:INSA_Toulouse/contenu/lab_practice/notebook/protocols/charac_recomb]]
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'''Results'''
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In the absence of recombinase or in recombination assays with Bxb1, the resulting colonies are white. Therefore, non-specific recombinases cannot switch the XOR gate. When XOR was put in the presence of FimE, the presence of red colonies validated the switch.
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However, only 5% of the plasmids were switched. Hence, the switch of XOR using FimE was not highly efficient. Several explanations can be made: i) low expression of FimE; ii) low activity of FimE in the tested buffer; iii) low activity of FimE on our designed gate. Improvement on FimE protocols or redesign of new sites could have been performed with our Michigan buddies but, unfortunately, time was running out.
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https://static.igem.org/mediawiki/2013/b/b3/X2.PNG
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We also performed a second switch, using the XOR gate that has been switched by FimE. We prepared the DNA from the XOR gate that has been previously switched by FimE (a red colony). This plasmid DNA was tested in the presence of PhiC31 recombinase. The picture below demonstrates that the second switch occurred: white colonies are now present.
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https://static.igem.org/mediawiki/2013/6/68/XOR2_result.PNG
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https://static.igem.org/mediawiki/2013/a/ae/Xor_2_swiche_par_Fim_E_avec_PhiC31.PNG
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  '''''Percent of red and white colony against reaction time of XOR switched by FimE with PhiC31 recombinases.'''''
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We also tried the switch with PhiC31 only. We used the original Siuti’s plasmid containing PhiC31, but no switch was obtained. The results might be due to low expression levels of PhiC31 that would lead to partial switch in in vitro conditions. A second explanation would be that the site we have designed is less efficient when the FimE recombinase has not yet performed the primary switch. More experiments are needed to verify PhiC31’s efficiency.
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===User Reviews===
 
===User Reviews===

Revision as of 00:54, 5 October 2013


Applications of BBa_K1132038

In vitro characterization of BBa_K1132036

The basic idea is to perform the switch with a cell lysis containing the recombinase and the plasmid containing the gate to be switched. After incubation, transformation of the plasmid containing the gate allows the quantification of switched versus non switched plasmids. Experiments have been performed as described in the protocol. http://2013.igem.org/Team:INSA_Toulouse/contenu/lab_practice/notebook/protocols/charac_recomb

Results

In the absence of recombinase or in recombination assays with Bxb1, the resulting colonies are white. Therefore, non-specific recombinases cannot switch the XOR gate. When XOR was put in the presence of FimE, the presence of red colonies validated the switch. However, only 5% of the plasmids were switched. Hence, the switch of XOR using FimE was not highly efficient. Several explanations can be made: i) low expression of FimE; ii) low activity of FimE in the tested buffer; iii) low activity of FimE on our designed gate. Improvement on FimE protocols or redesign of new sites could have been performed with our Michigan buddies but, unfortunately, time was running out.

X2.PNG

We also performed a second switch, using the XOR gate that has been switched by FimE. We prepared the DNA from the XOR gate that has been previously switched by FimE (a red colony). This plasmid DNA was tested in the presence of PhiC31 recombinase. The picture below demonstrates that the second switch occurred: white colonies are now present.

XOR2_result.PNG

Xor_2_swiche_par_Fim_E_avec_PhiC31.PNG 

 Percent of red and white colony against reaction time of XOR switched by FimE with PhiC31 recombinases.

We also tried the switch with PhiC31 only. We used the original Siuti’s plasmid containing PhiC31, but no switch was obtained. The results might be due to low expression levels of PhiC31 that would lead to partial switch in in vitro conditions. A second explanation would be that the site we have designed is less efficient when the FimE recombinase has not yet performed the primary switch. More experiments are needed to verify PhiC31’s efficiency.


User Reviews

UNIQba695253b1f75bb2-partinfo-00000000-QINU UNIQba695253b1f75bb2-partinfo-00000001-QINU