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Team:ITU MOBGAM Turkey/project
From 2013.igem.org
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As an iGEM team, our aim is to synthesize and secrete intrinsic factor (IF) protein in ileum by using E. coli. We determined our IF production and secretion location as ileum instead of stomach due to possible negative effects of low pH value in stomach. Because ileum is already serves as the location for binding of IF with B12 vitamin and absorption, choosing ileum will be advantageous. In addition to this, the possibility of occurrence of an autoimmune disease will be eliminated by providing E. coli its natural flora, which is ileum.
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Project Details
Part 2
In the first stage of the project, the production of intrinsic factor protein is aimed. For the production of IF, which is a protein composed of 417 amino acids encoded by 1254 nucleotides, gif gene will be designed according to the iGEM RFC standards and synthesized by GenScript Company as it will be contained in pUC57 cloning vector. In the second stage of the project, recombinant IF protein is aimed to be secreted into the extracellular matrix of E. coli cells. For this purpose, TYPE II secretion system signal sequence will be added to gif gene. To add the required signal sequence, promoter, ribosome binding site (RBS) and DNA fragments which include signal sequence will be synthesized by GeneScript Company. With the help of 3A union strategy, gif gene and synthesized DNA sequences will be joined and transferred into a new vector system. ImmunBlot will be used in order to identify the most appropriate signal sequence for the secretion of IF protein. In the last stage of the project, IF protein will be cloned into pGEX-6-1 vectors system and will be purified via GST-tag purification method. Pure GIF protein will be analyzed in order to determine the presence or absence of its complex with B12 vitamin based on the previous work of Wen and his colleagues (2000). During the project, translation of gif gene will be provided by a promoter that can be induced by bicarbonate. This will be achieved by using BBa_K872000 coded reference part that is found in the Registry of Biological part gene collection.
