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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, the colonies are white. As a result, non-specific recombinases cannot switch the AND gate.
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| − | https://static.igem.org/mediawiki/2013/e/ed/A2.PNG
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| − | We tested the AND gate with FimE. After incubation with FimE, the colonies are still white (this is normal!), therefore visual inspection of the switch was not possible. The only possibility was to sequence independent clones due to lack of time we did not have the results (see genotype section). We analyzed five independent colonies by sequencing, but none was switched. Several hypotheses can be made. First, the presence of the PhiC31 switching sequences may inhibit.
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| − | The AND was also tested with the Siuti’s construction parts containing PhiC31, but not switched has been obtained. 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=== |
Latest revision as of 03:33, 5 October 2013
User Reviews
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