Team:Slovenia/PROJECT/proof/studies/prod

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<h2>Cloning scheme</h2>
<h2>Cloning scheme</h2>
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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&nbsp;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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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&nbsp;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.<html> Parts used for production were: <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K323001"><span style="color: #810081;">BBa_323001</span></a><a href="/w/page/BBa_323001"></a>, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K323015"><span style="color: #0000ff;">BBa_323015</span></a>, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K323069"><span style="color: #0000ff;">BBa_323069</span></a>, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K323058"><span style="color: #0000ff;">BBa_323058</span></a>, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K323004"><span style="color: #0000ff;">BBa_323004</span></a>, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K323070"><span style="color: #0000ff;">BBa_323070</span></a>.</html>
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[[Image:SLOSPR_shema.png|thumb|center|700px]]
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<p><strong><span style="font-size: 16px; line-height: 25px;">Protein production</span></strong></p>
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<h2>Protein production</h2>
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Zinc fingers were produced in <em>E.coli</em> BL21(DE3)pLysS and purified with Ni-NTA column according to our protocols in <html><a href="http://2010.igem.org/Team:Slovenia/METHODS_and_PARTS/protocols">Methods &amp; Parts</a>. Their isolation and purity was confirmed with SDS-page and Western blot techniques. Secondary structure of proteins was determined with circular dichroism. Purified proteins were further used for <a href="http://2010.igem.org/Team:Slovenia/PROJECT/proof/studies/emsa">electrophoretic mobility shift assay</a> (EMSA) test and <span style="font-size: 115%;"><a href="http://2010.igem.org/Team:Slovenia/PROJECT/proof/studies/spr">surface plasmon resonance</a> (SPR) experiments.</html><br>
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Zinc fingers were produced according to our protocols in Methods &amp; Parts and their&nbsp;isolation and purity&nbsp;confirmed with SDS-page and Western blot techniques. With circular dichroism we confirmed the secondary structure of purified proteins.
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[[Image:SLOvsi.jpg|thumb|center|700px|'''SDS page of purified zinc fingers.''' Purified proteins were loaded on 10 % SDS gel and stained with Coommasie Blue stain. The arrows show the proteins of interest with their expected molecular weight, according to the molecular weight calculated with ProtParam tool.]]<br><br>
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[[Image:SLOvsi-western.jpg|thumb|center|700px|''Western blot confirmed isolated zinc fingers.''' We have been able to detect purified proteins with anti-his primary antibodies on Western blot. Arrows indicate each protein and its molecular weight.''']]<br><br>
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All six proteins were also tested with circular dichorism spectroscopy. Secondary structures were observed successfully:
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[[Image:CDvsiprot(Matej).jpg|thumb|center|700px|'''CD spectroscopy proved our proteins folds secondary structures.''' Secondary structure was determined with CD spectroscopy. From this figure we can see that most of the purified proteins refolded mainly in &alpha;-helical structure with small portion of random coil, except Gli wich has mostly &alpha;-helical structure.]]<br><br>
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Revision as of 17:24, 27 October 2010

Chuck Norris facts:

Production of binding proteins EMSA SPR BETA-GAL binding studies - Production of binding proteins



Contents


Cloning scheme

Before any in vitro 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 E. coli BL21(DE3)pLysS production strain. Zinc fingers were then purified using His tags and used in subsequent experiments. Parts used for production were: BBa_323001, BBa_323015, BBa_323069, BBa_323058, BBa_323004, BBa_323070.

SLOSPR shema.png

Protein production

Zinc fingers were produced in E.coli BL21(DE3)pLysS and purified with Ni-NTA column according to our protocols in Methods & Parts. Their isolation and purity was confirmed with SDS-page and Western blot techniques. Secondary structure of proteins was determined with circular dichroism. Purified proteins were further used for electrophoretic mobility shift assay (EMSA) test and surface plasmon resonance (SPR) experiments.

SDS page of purified zinc fingers. Purified proteins were loaded on 10 % SDS gel and stained with Coommasie Blue stain. The arrows show the proteins of interest with their expected molecular weight, according to the molecular weight calculated with ProtParam tool.


Western blot confirmed isolated zinc fingers. We have been able to detect purified proteins with anti-his primary antibodies on Western blot. Arrows indicate each protein and its molecular weight.


All six proteins were also tested with circular dichorism spectroscopy. Secondary structures were observed successfully:

CD spectroscopy proved our proteins folds secondary structures. Secondary structure was determined with CD spectroscopy. From this figure we can see that most of the purified proteins refolded mainly in α-helical structure with small portion of random coil, except Gli wich has mostly α-helical structure.