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Team:Evry/TeamSlovenia collaboration
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<center><h1><b>Characterization of parts from Team Slovenia 2012</b></h1></center> | <center><h1><b>Characterization of parts from Team Slovenia 2012</b></h1></center> | ||
| + | </br> | ||
| + | <p> | ||
| + | To test our chassis we decided to do a collaboration with a team working in vertebrate cells. | ||
| + | After the Regional Jamboree, and the presentation of <a href="https://2012.igem.org/Team:Slovenia">Team Slovenia</a> during the final, we asked them to test one of their construction. Especially constructions using TAL activator constructs (basement of their switch system). | ||
| + | </p> | ||
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| + | <p>So they send us part <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K782029">BBa_K782029</a> in the eukaryotic plasmid pCDNA3, and part <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K782065">BBa_K782065</a> in the eukaryotic plasmid pGL4.16. | ||
| + | </p> | ||
| - | + | <p>The first plasmid possesses the part <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K782065">BBa_K782065</a> which contains the TAL A effector and the VP16 core under the control of the constitutive CMV promoter | |
| - | + | </p> | |
| - | + | <p>The second plasmid possesses the part <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K782029">BBa_K782029</a> which contains 10 binding sites for TALA followed by the minimal promoter and the mCitrine reporter. | |
| + | </p></br> | ||
| - | The | + | <h2>The TAL A system in <i>Xenopus</i> embryos</h2> |
| + | </br> | ||
| + | <p>To test this system we first injected embryos with the plasmid <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K782029">BBa_K782029</a> which will express few mCitrine. | ||
| + | In a second hand both plasmids, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K782029">BBa_K782029</a> and <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K782065">BBa_K782065</a> were co-injected. The TALA VP16 expressed with the CMV promoter bind the 10x[A] sequence of the second plasmid and over-express mCitrine. | ||
| + | </p> | ||
| + | <center> | ||
| + | <img src="https://static.igem.org/mediawiki/2012/f/fc/Injection_mcitrineTeamSlovenia.png" width=600px> | ||
| + | </center> | ||
| + | </br> | ||
| + | <p> | ||
| + | We injected plasmids with rhodamine to be sure that embryos were injected. | ||
| + | The result show that with only the plasmid with the reporter under the minimal promoter, mCitrine is not expressed. But with the co-injection of this plasmid and the plasmid with TALA-VP16 under the control of the CMV promoter, mCitrine is expressed. It means that the switch works and the TALA-VP16 trigger the expression of mCitrie when TALA-VP16 bind the recognition site for TALA in the plasmid with mCitrine. | ||
| + | </br> | ||
| + | The mCitrine were still expressed 3 days after injection, and tadpoles were developing. | ||
| + | Details of this system is visible <a href="https://2012.igem.org/Team:Slovenia/TheSwitchDesignedTALregulators">here</a> (Team Slovenia page). | ||
| + | </p> | ||
| + | </br> | ||
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Latest revision as of 01:03, 27 October 2012
Characterization of parts from Team Slovenia 2012
To test our chassis we decided to do a collaboration with a team working in vertebrate cells. After the Regional Jamboree, and the presentation of Team Slovenia during the final, we asked them to test one of their construction. Especially constructions using TAL activator constructs (basement of their switch system).
So they send us part BBa_K782029 in the eukaryotic plasmid pCDNA3, and part BBa_K782065 in the eukaryotic plasmid pGL4.16.
The first plasmid possesses the part BBa_K782065 which contains the TAL A effector and the VP16 core under the control of the constitutive CMV promoter
The second plasmid possesses the part BBa_K782029 which contains 10 binding sites for TALA followed by the minimal promoter and the mCitrine reporter.
The TAL A system in Xenopus embryos
To test this system we first injected embryos with the plasmid BBa_K782029 which will express few mCitrine. In a second hand both plasmids, BBa_K782029 and BBa_K782065 were co-injected. The TALA VP16 expressed with the CMV promoter bind the 10x[A] sequence of the second plasmid and over-express mCitrine.
We injected plasmids with rhodamine to be sure that embryos were injected. The result show that with only the plasmid with the reporter under the minimal promoter, mCitrine is not expressed. But with the co-injection of this plasmid and the plasmid with TALA-VP16 under the control of the CMV promoter, mCitrine is expressed. It means that the switch works and the TALA-VP16 trigger the expression of mCitrie when TALA-VP16 bind the recognition site for TALA in the plasmid with mCitrine. The mCitrine were still expressed 3 days after injection, and tadpoles were developing. Details of this system is visible here (Team Slovenia page).
