Difference between revisions of "Team:Georgia State/Description"

 
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  <a href="https://2015.igem.org/Team:Georgia_State/Attributions">Attributions</a>
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  <a href="https://2015.igem.org/Team:Georgia_State/Project#Thing1">Mambalgin</a>
 
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  <a href="https://2015.igem.org/Team:Georgia_State/Practices">Practices</a>
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<a href="https://2015.igem.org/Team:Georgia_State/Collaborations">Collaborations</a>
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<a href="https://2015.igem.org/Team:Georgia_State/Description">Description</a>
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<a href="https://2015.igem.org/Team:Georgia_State/Design">Design</a>
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<li> <a href="https://2015.igem.org/Team:Georgia_State/Notebook">Notebook</a>
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<a href="https://2015.igem.org/Team:Georgia_State/ImageGallery">Image Gallery</a>
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<a href="https://2015.igem.org/Team:Georgia_State/Practices#Thing4">Atlanta Botanical Gardens</a>
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<a href="https://2015.igem.org/Team:Georgia_State/Collaborations#Thing2">Project JBroth</a>
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<li> <a href="https://2015.igem.org/Team:Georgia_State/Safety">Safety</a>
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                         <h3 class="h4" style="text-align: center;color: white;">Our Parts</h3>
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                         <h3 class="h4" style="text-align: center;color: black;"><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1110003">Mambalgin </h3>
 
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                         <p class="lead"  style="color: white;" ><a class="tipped" data-title="Abstract" data-tipper-options='{"direction":"top"}'><b>The parts we designed are the best parts in all of iGEM. Our parts are designed to the highest quality. Trust us. No better quality can be found. IDT helped us out a good bit, they're cool. Most our parts have coding sequences. Don't worry about the parts that don't have coding sequences. </p>
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                         <p class="lead"  style="color: black;" ><a class="tipped" data-title="Abstract" data-tipper-options='{"direction":"top"}'><b>For the design, we took the coding sequence for mambalgin, removed the TAG stop codon, and added RFC 25 prefix and suffix in Snapgene software to visualize. RFC 25 was used because we were anticipating the use of this part in creating fusion proteins. Additional nucleotides were added before the biobrick prefix and after the biobrick suffix to enable more efficient restriction enzyme cutting at EcoRI and PstI sites. The mambalgin construct was then ordered from IDT and ligated into pSB1C3 backbone. Using the pGAPzα expression system provided by ThermoFisher Scientific, the GSU team has been working to express the protein in Pichia Pastoris. The pGAPZα vector includes the constitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter, an alpha secretory signal peptide, and Myc and 6x His epitopes. The MCS, between the alpha secretion signal and epitopes, consists of restriction sites which will enable insertion of mambalgin. In-frame insertion into the MCS of pGAPza will be possible after using PCR to modify the construct - removing the RFC 25 prefix and suffix and adding restriction sites and nucleotides. </p>
 
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Latest revision as of 17:56, 8 November 2015

IGEM Team:GSU


Design


Mambalgin

For the design, we took the coding sequence for mambalgin, removed the TAG stop codon, and added RFC 25 prefix and suffix in Snapgene software to visualize. RFC 25 was used because we were anticipating the use of this part in creating fusion proteins. Additional nucleotides were added before the biobrick prefix and after the biobrick suffix to enable more efficient restriction enzyme cutting at EcoRI and PstI sites. The mambalgin construct was then ordered from IDT and ligated into pSB1C3 backbone. Using the pGAPzα expression system provided by ThermoFisher Scientific, the GSU team has been working to express the protein in Pichia Pastoris. The pGAPZα vector includes the constitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter, an alpha secretory signal peptide, and Myc and 6x His epitopes. The MCS, between the alpha secretion signal and epitopes, consists of restriction sites which will enable insertion of mambalgin. In-frame insertion into the MCS of pGAPza will be possible after using PCR to modify the construct - removing the RFC 25 prefix and suffix and adding restriction sites and nucleotides.