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| − | ===In vitro characterization ===
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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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| − | 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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| − | This part can be considered as a good amelioration of the RFP reporter system. Reporter systems can be controlled either by regular positive or negative regulators. Such regulation systems obey to biological rules and are modulated between to limit values. The logic gates obey a second type of rule, closer to the electronic description of the 0 and 1 (Off and On) states. Therefore, the switch are less prone to physiological states than regulatable promoters. Furthermore, we also demonstrated that the switch are permanent and can be genetically transmitted to the offspring. This also constitutes a new system for determining at any time any occurring event that can further disappear. The switch would serve as a tracer. Last, as these switches obey Boolean logic operations, detection of independent, time separated events can also be achieved. A full description of the logic gates is present on our [http://2013.igem.org/Team:INSA_Toulouse/contenu/lab_practice/results/logic_gates Wiki].
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| | ===User Reviews=== | | ===User Reviews=== |
UNIQ967bb71e595df219-partinfo-00000000-QINU
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