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Team:Potsdam Bioware/Project/Details
From 2011.igem.org
Details
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Microviridin
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Phage Display
Phage Display is an efficient tool for selecting protein or peptides with specific binding properties from a large recombinant library. This proteins are represented on the surface of bacteriophages. This enables the coupling of phenotype and stable packaged genotype because the proteins which form the phage including the proteins of interest are coded in its genome. To identify mdnA-varieties which act as protease inhibitors, a mdnA-library containing randomly mutated mdnA was created. For this purpose the phagemid vector pPARW089 was produced. This vector contains a plasmid origin of replication, so they can be amplified like plasmids additionally it contains a f1 ori which enables the packaging of single strand DNA into phages. The vector also contains the whole mdn-cluster which is needed to produce the mdnA peptide. Cloning of the mutated mdnA genes into the phagemid generates a mdnA-gene III- fusion gene. Between mdnA and gene III a myc-tag for detection is located. The successful expression of the mdnA-myc-geneIII fusion protein on the surface of the phage was determined by ELISA test using anti-myc-antibodies after transforming E. coli cells and purifying the produced phages. The next step was the performance of a phage display. To test the fundamental suitability of this screening method, phages representing mdnA on their surface and phages not representing mdnA in a ratio of one to one were incubated with immobilised trypsin which is known as a target of mdnA. After one panning round a marked concentrating of phages carrying mdnA was recognized. For the screening of the mdnA-library the phage display vector pPARWIII was created….
In Vivo Selection
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Modelling
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