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| − | == '''In vivo characterization''' ==
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| − | <p class="texte">The principle of this characterization is the co-transformation of the gate and the recombinase. The gate construct (BBa_K1132032) is on chloramphenicol and the Bxb1 construct (BBa_K1132027) is on kanamycine, we used 20 ng of each plasmid to transform E. coli and let it growth on chloramphenicol and kanamycine. Some colonies were red after transformation. However, in these conditions, the genotype of the gate (presence of the switch) is hard to verify due to the presence of two plasmids in the cotransformed strain. We therefore resorted to deeper characterization with fluorescence measurements. The positive control is a plasmid expressing constitutively RFP and the negative control is the co-transformation of XOR with a plasmid not containing any recombinases but on the same antibiotic resistance. </p>
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| − | https://static.igem.org/mediawiki/2013/3/33/Graph2.png
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| − | https://static.igem.org/mediawiki/2013/b/bd/Legende_graph.png
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| − | <div class="clear"></div>
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| − | <p class="texte">On the above graph, there is a clear difference when XOR is cotransformed with Bxb1 contrarily to the negative control .A deeper analysis of the fluorescence of the strain demonstrated that the level of fluorescence of the negative control is actually just diffusion of light. This result confirms the switch. The low fluorescence of our strains compared to the positive control could be due to the presence of a weak promoter in our gates and/or the long distance between the promoter and the coding sequence.
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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.[[http://2013.igem.org/Team:INSA_Toulouse/contenu/lab_practice/notebook/protocols/charac_recomb]]
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| − | '''''Phenotype study (visual inspection or fluorimetric assays)'''''
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| − | In the absence of recombinase or in recombination assays with FimE, the resulting colonies are white. Therefore, the XOR gate does not present any transcriptional leakage and non-specific recombinases cannot switch the door. When XOR was put in the presence of Bxb1, the switch occurred as attested by the presence of numerous red colonies (RFP expression).
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| − | https://static.igem.org/mediawiki/2013/7/73/X1.PNG
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| − | To test the time dependency of the reaction, the reaction mixture incubated was analyzed at different time from 15 min to 2.5 hours
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| − | https://static.igem.org/mediawiki/2013/9/9c/Xor1-_Bxb1_%2815min_-2%2C5h%29.PNG
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| − | From the graph above, one can see that switching occurs after 15 min. After 2 hours reaction, over 90 % of XOR gates were switched and the percentage of XOR1 gates switched from 2 hours to 5 hours reaction is almost constant (not shown).
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| − | https://static.igem.org/mediawiki/2013/d/d6/Xor1_-Dnase.PNG
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| − | In the in vitro protocol, DNAses were not eliminated. Therefore, longer times reactions also means that plasmids containing the gates can also be degraded. This could explain why, when incubation lasted longer than 2 hours, the number of colonies dropped as shown in the following graph.
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| − | We also tried to switch XOR with the two recombinases. The XOR gate was tested with the Dual Controller Plasmid provided by Bonnet (KC529324) and containing Tp901.1 and Bxb1. No switch was ever obtained. This negative result might be due to low expression levels of any of the two recombinases that would lead to partial switch in in vitro conditions. Further experiments will have to be performed to understand this phenomenon.
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| − | '''''Genotype study'''''
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| − | We analyzed XOR switched plasmids by sequencing. The sequence of switched plasmid corresponds to the expected switches.
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| − | https://static.igem.org/mediawiki/2013/thumb/6/60/Switch_XOR_1.png/676px-Switch_XOR_1.png
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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=== |
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