Team:UGent/Experiments
From 2013.igem.org
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[[File:UGent_2013_Datsenko-Wanner.jpg|thumb|450px|center|Datsenko-Wanner, 2000]] | [[File:UGent_2013_Datsenko-Wanner.jpg|thumb|450px|center|Datsenko-Wanner, 2000]] | ||
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- | <td align="center" width="500px"><img src="https://static.igem.org/mediawiki/2013/4/44/UGent_2013_Exp1.jpg" width=" | + | <td align="center" width="500px"><img src="https://static.igem.org/mediawiki/2013/4/44/UGent_2013_Exp1.jpg" width="450"> |
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<center><img src="https://static.igem.org/mediawiki/2013/4/44/UGent_2013_Exp1.jpg" width="700"></center> <br> | <center><img src="https://static.igem.org/mediawiki/2013/4/44/UGent_2013_Exp1.jpg" width="700"></center> <br> | ||
<A HREF="https://static.igem.org/mediawiki/2013/4/46/UGent_2013_Experiment_2.pdf" target="_blank">Protocol experiment 2</A> | <A HREF="https://static.igem.org/mediawiki/2013/4/46/UGent_2013_Experiment_2.pdf" target="_blank">Protocol experiment 2</A> | ||
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<h2> Experiment 3 </h2> | <h2> Experiment 3 </h2> | ||
<p> Once we have constructed the plasmid with ccdB and a T7 promoter, it has to be transferred into the cells in which CIChE will be performed. This “transformation” (the process of putting DNA into a cell) will be carried out in experiment 3.</p> | <p> Once we have constructed the plasmid with ccdB and a T7 promoter, it has to be transferred into the cells in which CIChE will be performed. This “transformation” (the process of putting DNA into a cell) will be carried out in experiment 3.</p> | ||
+ | <center><img src="https://static.igem.org/mediawiki/2013/9/91/UGent_2013_Exp3.jpg" width="700"></center> <br> | ||
<A HREF="https://static.igem.org/mediawiki/2013/9/91/UGent_2013_Experiment_3_Construction_CIChE_strains.pdf" target="_blank">Protocol experiment 3</A> | <A HREF="https://static.igem.org/mediawiki/2013/9/91/UGent_2013_Experiment_3_Construction_CIChE_strains.pdf" target="_blank">Protocol experiment 3</A> | ||
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<h2> Experiment 4 </h2> | <h2> Experiment 4 </h2> | ||
<p>Strains constructed in experiment 3 will be used to perform CIChE. Tandem gene replication of reporter protein GFP will be induced by replicating the antitoxin ccdA as a response on titration of the toxin ccdB. Titration of ccdB under inducible T7-promoter will be accomplished by different levels of IPTG [0.01 mM – 0.5mM] and different plasmid copy numbers [p5, p10 and p20]. </p> | <p>Strains constructed in experiment 3 will be used to perform CIChE. Tandem gene replication of reporter protein GFP will be induced by replicating the antitoxin ccdA as a response on titration of the toxin ccdB. Titration of ccdB under inducible T7-promoter will be accomplished by different levels of IPTG [0.01 mM – 0.5mM] and different plasmid copy numbers [p5, p10 and p20]. </p> |
Revision as of 14:17, 1 October 2013
ExperimentsTo test our idea we are conducting 6 experiments. These are described below. Experiment 1Experiment 1 is the knock-in (KI) of the construct containing ccdA, GFP and the homologous regions (this was constructed beforehand). This will be done by using the method of Datsenko & Wanner [PNAS 2000], based on homologous recombination. The principle of the KO/KI method is depicted below:
Experiment 2Next is finding a way to administer the toxin to the cells. This will be done by putting the gene coding for the toxin on a plasmid (a linear piece of DNA) and transferring this plasmid into the bacterial cell. In experiment 2 a plasmid containing ccdB (toxin) under control of a T7 promotor will be constructed. The T7 promoter allows us to control the expression of ccdB by regulating the amount of IPTG added to the cells. ![]() Protocol experiment 2 Experiment 3Once we have constructed the plasmid with ccdB and a T7 promoter, it has to be transferred into the cells in which CIChE will be performed. This “transformation” (the process of putting DNA into a cell) will be carried out in experiment 3. ![]() Protocol experiment 3 Experiment 4Strains constructed in experiment 3 will be used to perform CIChE. Tandem gene replication of reporter protein GFP will be induced by replicating the antitoxin ccdA as a response on titration of the toxin ccdB. Titration of ccdB under inducible T7-promoter will be accomplished by different levels of IPTG [0.01 mM – 0.5mM] and different plasmid copy numbers [p5, p10 and p20]. Protocol experiment 4Experiment 5Experiment 6In this experiment, a new part, called BBa_K1105000, will be constructed by cloning Laciq-T7ccdB with standard Biobrick prefix and suffix in pSB1C3. Laciq-T7ccdB is derived from the plasmid p10-LacIq-T7ccdB, which originally comes from a mini F-plasmid positive strains (such as E. coli F+). The function of this part is to produce CcdB (in control of a T7 promotor), which interferes with the topoisomerase unit gyrA. Protocol experiment 6
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