Team:UGent/Experiments
From 2013.igem.org
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- | <p> Experiment 1 is the <b>knock-in</b> (KI) of the construct containing <i>ccdA</i>, <i>gfp</i> and the homologous regions (this was constructed beforehand). This will be done by using the method of <A HREF="https://static.igem.org/mediawiki/2013/1/18/UGent_2013_Datsenko-Wanner.jpg" target="_blank">Datsenko & Wanner [PNAS 2000]</A>, based on homologous recombination. The principle of the KO/KI method is depicted | + | <p> Experiment 1 is the <b>knock-in</b> (KI) of the construct containing <i>ccdA</i>, <i>gfp</i> and the homologous regions (this was constructed beforehand). This will be done by using the method of <A HREF="https://static.igem.org/mediawiki/2013/1/18/UGent_2013_Datsenko-Wanner.jpg" target="_blank">Datsenko & Wanner [PNAS 2000]</A>, based on homologous recombination. The principle of the KO/KI method is depicted left.</p> |
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+ | <p><A HREF="https://static.igem.org/mediawiki/2013/f/ff/UGent_2013_Experiment_1_KI_ccdA-Pmb1-GFP.pdf" target="_blank">Protocol experiment 1</A> | ||
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- | <p> Next 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 <i>ccdB</i> (toxin) under control of a T7 | + | <p> Next 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 <i>ccdB</i> (toxin) under control of a T7 promoter will be constructed. The T7 promoter allows us to control the expression of <i>ccdB</i> by regulating the amount of IPTG added to the cells.<br><br> |
+ | During this experiment we encountered some unexpected mutations and used a reverse mutation protocol, based on Gibson Assembly, to restore the original plasmid sequence. </p> | ||
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- | <A HREF="https://static.igem.org/mediawiki/2013/4/46/UGent_2013_Experiment_2.pdf" target="_blank">Protocol experiment 2</A> | + | <br><p><A HREF="https://static.igem.org/mediawiki/2013/4/46/UGent_2013_Experiment_2.pdf" target="_blank">Protocol experiment 2</A> |
+ | <br><p><A HREF="https://static.igem.org/mediawiki/2013/c/c0/UGent_2013_Protocol_Back_mutation_using_Gibson_Assembly.pdf" target="_blank">Protocol for Back Mutation (using Gibson Assembly)</A></p> | ||
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<h2> Experiment 3 </h2> | <h2> Experiment 3 </h2> | ||
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- | <A HREF="https://static.igem.org/mediawiki/2013/9/91/UGent_2013_Experiment_3_Construction_CIChE_strains.pdf" target="_blank">Protocol experiment 3</A> | + | <br> |
+ | <p><A HREF="https://static.igem.org/mediawiki/2013/9/91/UGent_2013_Experiment_3_Construction_CIChE_strains.pdf" target="_blank">Protocol experiment 3</A> | ||
+ | </p> | ||
<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>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]. |
- | <A HREF="https://static.igem.org/mediawiki/2013/c/ca/UGent_2013_Experiment_4_CIChE_using_ccdAccdB.pdf" target="_blank">Protocol experiment 4</A> | + | <br> |
+ | <br><A HREF="https://static.igem.org/mediawiki/2013/c/ca/UGent_2013_Experiment_4_CIChE_using_ccdAccdB.pdf" target="_blank">Protocol experiment 4</A> </p> | ||
<h2> Experiment 5 </h2> | <h2> Experiment 5 </h2> | ||
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We used two protocols for this experiment. In the first version bacteria are grown for approximately 9 hours inside the FLUOstar and fluorescence and OD are measured each five minutes. The second protocol is less time consuming and consists of growing our bacteria in microtiter plates during 4 hours at 37°C (not in FLUOstar) after which fluorescence and OD are measured one time each inside the FLUOstar. | We used two protocols for this experiment. In the first version bacteria are grown for approximately 9 hours inside the FLUOstar and fluorescence and OD are measured each five minutes. The second protocol is less time consuming and consists of growing our bacteria in microtiter plates during 4 hours at 37°C (not in FLUOstar) after which fluorescence and OD are measured one time each inside the FLUOstar. | ||
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+ | <br><A HREF="https://static.igem.org/mediawiki/2013/7/7d/UGent_2013_Protocol_Experiment_5.1.pdf" target="_blank">Protocol experiment 5.1</A> | ||
+ | <br><A HREF="https://static.igem.org/mediawiki/2013/0/09/UGent_2013_Protocol_Experiment_5.2.pdf" target="_blank">Protocol experiment 5.2</A> | ||
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<h2> Experiment 6 </h2> | <h2> Experiment 6 </h2> | ||
- | <p> In this experiment, a | + | <p> In this experiment, a <A HREF="https://2013.igem.org/Team:UGent/Parts" target="_blank">new part</A>, called <A HREF="http://parts.igem.org/Part:BBa_K1105000" target="_blank">BBa_K1105000</A>, will be constructed by cloning LacIq-T7<i>ccdB</i> with standard Biobrick prefix and suffix in pSB1C3. Laciq-T7<i>ccdB</i> is derived from the plasmid p10-LacIq-T7<i>ccdB</i>, which originally |
- | comes from a mini F-plasmid positive strains (such as <i>E. coli</i> F+). The function of this part is to produce CcdB (in control of a T7 promotor), which interferes with the topoisomerase unit | + | comes from a mini F-plasmid positive strains (such as <i>E. coli</i> F+). The function of this part is to produce CcdB (in control of a T7 promotor), which interferes with the topoisomerase unit GyrA.<br><br> |
- | + | During this experiment we encountered some unexpected mutations and used a reverse mutation protocol, based on Gibson Assembly, to restore the original plasmid sequence. | |
- | <A HREF="https://static.igem.org/mediawiki/2013/1/15/UGent_2013_Experiment_6_Cloning_pSB1C3-T7ccdB.pdf" target="_blank">Protocol experiment 6</A> | + | <br> |
- | + | <br><A HREF="https://static.igem.org/mediawiki/2013/1/15/UGent_2013_Experiment_6_Cloning_pSB1C3-T7ccdB.pdf" target="_blank">Protocol experiment 6</A> | |
+ | <br><A HREF="https://static.igem.org/mediawiki/2013/c/c0/UGent_2013_Protocol_Back_mutation_using_Gibson_Assembly.pdf" target="_blank">Protocol for Back Mutation (using Gibson Assembly)</A></p> | ||
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Latest revision as of 01:47, 5 October 2013
ExperimentsTo test our idea we are conducting 6 experiments. These are described below. Experiment 1
Experiment 2
Protocol for Back Mutation (using Gibson Assembly) 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].
Experiment 5Through experiment 4 we obtained strains that underwent different levels of chromosomal evolution and should contain different construct copy numbers. In experiment 5 we will assess the number of duplications by growing our colonies in microtiter plates and measuring fluorescence intensity and optical density (OD) inside the FLUOstar OPTIMA (BMG LABTECH). Bacteria with higher construct copy numbers also contain a higher number of gfp copies, resulting in higher fluorescence intensities.
Experiment 6 In 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.
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