Team:Utah State/Results

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<div style="position: absolute; top: 3520px;"><img src="https://static.igem.org/mediawiki/2012/8/88/Wheresspidey2.png" onMouseOver="this.src='https://static.igem.org/mediawiki/2012/9/92/SpiderFacts7.png'" onMouseOut="this.src='https://static.igem.org/mediawiki/2012/8/88/Wheresspidey2.png'"></div>
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<br><a name="DataSection"><h1 style="width: 100%; margin-left: 0px; margin-right: 20px; text-align: left; font-family:Arial, Helvetica, sans-serif; font-weight: normal; border-bottom: 2px solid #000000;" class="table-main">
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Data Section
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<br><a name="HowOurSystemWorks"><h1 style="width: 100%; margin-left: 0px; margin-right: 20px; text-align: left; font-family:Arial, Helvetica, sans-serif; font-weight: normal; border-bottom: 2px solid #C0C0C0;" class="table-main">
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How Our System Works
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<p align="center"><img src="https://static.igem.org/mediawiki/2012/b/b6/USU_HowOurSystemWorks2.PNG"></p>
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<br><a name="FavoriteParts"><h1 style="width: 100%; margin-left: 0px; margin-right: 20px; text-align: left; font-family:Arial, Helvetica, sans-serif; font-weight: normal; border-bottom: 2px solid #C0C0C0;" class="table-main">
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Data For Our Favorite New Parts
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<li><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K844016">Main page</a> - <b>Spider Silk Generator, BBa_K844016</b>: Contains a lac/IPTG promoter and 4x spider silk unit and a His-tag for purification. This part is fully functional (proved by protein gel) and is able to generate spider silk.
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<li><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K844000">Main page</a> - <b>10x Histidine (10x His)-Tag with double stop codon, BBa_K844000</b>: This part is fully functional as spider silk can be purified using a nickel column.
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<li><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K844015">Main page</a> - <b>lac/IPTG inducible Spider Silk 1x “F” Subunit fused GFP, BBa_K844015</b>: This part is a fully functional spider silk-GFP fusion protein as demonstrated by GFP flourescence.
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Data For Pre-existing Parts
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<li><a href="http://partsregistry.org/Part:BBa_K208010:Experience#User_Reviews
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">Experience</a> – <b>BBa_K208010, Lac Promoter and RBS</b> (Utah State iGEM 2009): This part was successfully able express of spider silk and spider silk-GFP fusion protein.
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<li><a href="http://partsregistry.org/Part:BBa_K208000:Experience#User_Reviews
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">Experience</a> – <b>BBa_K208000, GFP</b> (Utah State iGEM 2009): This part was successfully fused to a “F” spider silk subunit and was also successfully expressed.
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<br><a name="AlsoCharacterized"><h1 style="width: 100%; margin-left: 0px; margin-right: 20px; text-align: left; font-family:Arial, Helvetica, sans-serif; font-weight: normal; border-bottom: 2px solid #C0C0C0;" class="table-main">
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We've Also Characterized The Following Parts
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<li><a href="http://partsregistry.org/Part:BBa_K844008">Main page</a> - <b>Spider Silk 1x Subunit “B” (Balanced tRNA codon optimized) with Met (ATG) added, BBa_K844008</b> : Part works in BBa_K844016.
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<li><a href="http://partsregistry.org/Part:BBa_K844004">Main page</a> - <b>Spider silk 3x Subunit “B” (Balanced tRNA codon optimized), BBa_K844004:</b> Works in BBa_K844016. This part is optimized based on the native “W” construct, BBa_K844002.
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<li><a href="http://partsregistry.org/Part:BBa_K844007">Main page</a> – <b>Spider silk 1x Subunit “F” (Fewest tRNA codon optimized) with Met (ATG) added, BBa_K844007</b> : Works in BBa_K844015. This part is optimized based on native “W” construct, BBa_K844002.
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         Cloning
         Cloning
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Green fluorescent protein (GFP) has been used by various iGEM teams to demonstrate expression and functionality of a BioBrick system. In order to demonstrate that the expression of a BioBrick spider silk gene (F1) is possible in E.coli a single spider silk gene was tagged with GFP at the C terminus to demonstrate silk protein protein expression. The GFP that was chosen for this study was taken from Utah State iGEM 2009 (<a href= "http://partsregistry.org/wiki/index.php?title=Part:BBa_K208000"> BBa_K208000 </a>) as it demonstrated high levels of GFP expression. This GFP protein has an excitation wavelength of 395nm and an emission wavelength of 509nm. The lac promoter and ribosome binding site (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K208010"> BBa_K208010 </a>) used in this system was also taken from Utah State iGEM 2009. A plasmid map demonstrating this construct is shown below and this plasmid was transformed into DH5α.
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Green fluorescent protein (GFP) has been used by various iGEM teams to demonstrate expression and functionality of a BioBrick system. In order to demonstrate that the expression of a BioBrick spider silk gene (F1) is possible in E.coli a single spider silk gene was tagged with GFP at the C terminus to demonstrate silk protein expression. The GFP that was chosen for this study was taken from Utah State iGEM 2009 (<a href= "http://partsregistry.org/wiki/index.php?title=Part:BBa_K208000"> BBa_K208000 </a>) as it demonstrated high levels of GFP expression. This GFP protein has an excitation wavelength of 395nm and an emission wavelength of 509nm. The lac promoter and ribosome binding site (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K208010"> BBa_K208010 </a>) used in this system was also taken from Utah State iGEM 2009. A plasmid map demonstrating this construct is shown below and this plasmid was transformed into DH5α.
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        Protein Expression
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Protein Expression
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This 10x-Histidine tag (<a href= "http://partsregistry.org/Part:BBa_K844000">BBa_K844000</a>) was used to isolate and analyze spider silk protein produced by the 2012 Utah_State iGEM Team. Below is a Coomassie stained SDS PAGE gel showing the spider silk protein (~25.4 kDa) that was purified using a Nickel affinity resin column. The protein in the lane is nearly pure, with only minor bands at 24 kDa and 15 kDa as contaminants. The 10x-His tag aids in producing highly pure protein samples as it binds more tightly to the column, allowing higher concentration wash steps to be used to remove contaminants from the sample. Also below is a Western blot utilizing an antibody that binds specifically to histidine tags, showing a spider silk protein band at the correct molecular weight (~25.4 kDa). This demonstrates that the 10x-histidine tag is functioning and is compatible with staining and antibody techniques used with 6x-histidine tags.
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Coomassie blue stained SDS PAGE gel showing highly pure spider silk sample. Protein expressed is <a href= "http://partsregistry.org/Part:BBa_K844016">BBa_K844016</a>. The first lane contains the cell lysate sample. The second lane is the flowthrough from the column; note the absence of spider silk band from this flowthrough, indicating the high affinity of the 10x-His tag. The third lane is the eluted spider silk band, with only very minor contaminating bands. The last lane is the Bio-Rad Precision Plus Dual Color Protein Standard, with protein sizes indicated in kDa.
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Western Blot of Spider Silk Protein. Protein expressed is <a href="http://partsregistry.org/Part:BBa_K844016">BBa_K844016</a>. Control lane is <i>E. coli</i> DH5a cells without spider silk construct. Marker lane is Bio-Rad Precision Plus Dual Color Protein Standard. Primary antibody binds specifically to histidine tags. Staining was done with alkaline phosphatase attached to the secondary antibody.
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         Silk Production
         Silk Production
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