Team:Georgia State/project/overview

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Latest revision as of 00:04, 26 September 2013

Georgia State Wiki

   The primary purpose of our project is to modify the class of pGAPZα expression vectors according to iGEM standards to allow for expression and purification of proteins from an inducible yeast system. The glyceraldehyde-3-phosphate dehydrogenase (pGAP) promoter shuttle vector has been used in the methylotrophic yeast, Pichia pastoris, to express high levels of recombinant proteins (Waterham et al., 1997). The pGAPZα expression system allows for methanol-inducible expression of proteins with appropriate eukaryotic post-translational modifications. Additionally, the pGAPZα expression system allows for controlled secretion of a protein, making the purification of a protein relatively simple.

   Using the pGAPZα expression system, the GSU iGEM team has been working to express and purify mambalgin, a protein component of the venom of Dendroaspis polylepis, better known as the Black Mamba. The mambalgin peptide is a powerful analgesic that directly blocks pain transmission in the peripheral nervous system (Diochot et al, 2012) by targeting acid-sensing ion channels within nociceptors beneath the epidermis . Because mambalgin acts on pain receptors within the skin rather than on opioid receptors in the brain, this peptide has great potential as a medication for pain treatment that is non-addicting and non-habit forming. Furthermore, recombinant purification of mambalgin could assist in developing anti-venom without the attendant risk of harvesting venom directly from snakes.

   By the end of this competition period, we hope to have modified pGAPzα A, B, and C expression vectors to allow for easy insertion of any iGEM BioBricks in frame with the secretion factor and myc epitope and His tags which will allow for inducible production, eukaryotic processing, and simplified purification of any desired proteins. Our ultimate goal is to use this system to express and purify mambalgin, which may then be developed as a potent analgesic and as a potential antigen to produce black mamba antivenom.

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-Our project builds on previous years’ research and experiments working with the pGAPZαA, B, and C. We have been working towards standardizing all three vectors according to iGEM standards (MCS standards) in order to use these vectors to express genes through the Pichia Pastoris system. So far we were able to standardize pGAPZαA and express the Red Florescent Protein (RFP). We would like to standardize the B and C vectors as well, due to their varying reading frames. These vectors will be used in the Pichia Pastories
-The final goal of our new project is to be able to show capability of expressing and purifying a protein synthesized from the Black Mamba (Dendroaspis polylepis) venom. Following expression, we will be trying to elicit an immune response in small rodents (rats or rabbits). The lab responsible for the synthesis of the protein was able to test it only on the peripheral nervous system (PNS) and show an effect. We would like to be able to test the protein’s function in the central nervous system with the hope of showing a similar effect to that of the PNS. To achieve this we would collaborate with an additional lab on the Georgia State University campus, that works with rodents.
+&nbsp;&nbsp;&nbsp;The primary purpose of our project is to modify the class of pGAPZα expression vectors according to iGEM standards to allow for expression and purification of proteins from an inducible yeast system. The glyceraldehyde-3-phosphate dehydrogenase (pGAP) promoter shuttle vector has been used in the methylotrophic yeast, <i>Pichia pastoris</i>, to express high levels of recombinant proteins (Waterham et al., 1997). The pGAPZα expression system allows for methanol-inducible expression of proteins with appropriate eukaryotic post-translational modifications. Additionally, the pGAPZα expression system allows for controlled secretion of a protein, making the purification of a protein relatively simple.
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+&nbsp;&nbsp;&nbsp;Using the pGAPZα expression system, the GSU iGEM team has been working to express and purify mambalgin, a protein component of the venom of <i>Dendroaspis polylepis</i>, better known as the Black Mamba. The mambalgin peptide is a powerful analgesic that directly blocks pain transmission in the peripheral nervous system (Diochot et al, 2012) by targeting acid-sensing ion channels within nociceptors beneath the epidermis . Because mambalgin acts on pain receptors within the skin rather than on opioid receptors in the brain, this peptide has great potential as a medication for pain treatment that is non-addicting and non-habit forming. Furthermore, recombinant purification of mambalgin could assist in developing anti-venom without the attendant risk of harvesting venom directly from snakes.
+<br /><br />
+&nbsp;&nbsp;&nbsp;By the end of this competition period, we hope to have modified pGAPzα A, B, and C expression vectors to allow for easy insertion of any iGEM BioBricks in frame with the secretion factor and <i>myc</i> epitope and His tags which will allow for inducible production, eukaryotic processing, and simplified purification of any desired proteins.  Our ultimate goal is to use this system to express and purify mambalgin, which may then be developed as a potent analgesic and as a potential antigen to produce black mamba antivenom.
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