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Team:Slovenia/PROJECT/proof/studies/prod
From 2010.igem.org
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| + | <p><strong><span style="font-size: 16px; line-height: 25px;">Cloning scheme</span></strong></p> | ||
| + | <p> </p> | ||
| + | Before any <em>in vitro</em> characterisation, DNA-binding proteins had to be obtained. Zinc fingers were firstly tri-point ligated with a particular split GFP and T7 promoter and T7 terminator sequence were added afterwards. T7 promoter enables high production of proteins in<em> E. coli </em>BL21(DE3)pLysS production strain. Zinc fingers were then purified using His tags and used in subsequent experiments. Parts used for production were: [http://partsregistry.org/wiki/index.php?title=Part:BBa_K323001 BBa_323001], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K323015 BBa_323015], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K323069 BBa_323069], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K323058 BBa_323058], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K323004 BBa_323004], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K323070 BBa_323070]. | ||
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| - | + | [[Image:SLOSPR_shema.png|thumb|center|700px]] | |
<p> </p> | <p> </p> | ||
| - | + | <p><strong><span style="font-size: 16px; line-height: 25px;">Protein production</span></strong></p> | |
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| + | Zinc fingers were produced according to our protocols in Methods & Parts and their isolation and purity confirmed with SDS-page and Western blot techniques. With circular dichroism we confirmed the secondary structure of purified proteins. | ||
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<p> </p> | <p> </p> | ||
| - | [[Image: | + | [[Image:SLOvsi.jpg|thumb|center|700px]] |
<p> </p> | <p> </p> | ||
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Revision as of 12:45, 27 October 2010
