Team:HokkaidoU Japan/Design

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Design

INSERT 1

This part is for inhibiting INSERT 2, 3, or 4. This composite part is made up of constitutive promoter, RBS, LacI inhibitor coding sequence, and double terminator. When translated, it produces LacI inhibitors. This result in inhibiting Plac promoters in INSERT 2, 3, and 4 when there is no IPTG, whereas it release Plac promoter when there is IPTG.

INSERT 2 BBa_K2414002, BBa_K2414003

This composite part was designed to make circularized Phytase. When there is no LacI inhibition, it translates Phytase, with a linker, a SAP (self assembling peptide,) and a cysteine module (GGGS) on both N and C termini. In addition, there is a signal peptide on N terminus (for making correct conformation), and His-Tag on C terminus (for purification). This circularized Phytase is the main target of our project, because we can assay how circularized Phytase, whose circularization is made from SAPs’ hydrogen bond and cysteine modules’ disulfide bond, and normal Phytase are different from each other in terms of, for example, heat resistance and activity as enzyme.

INSERT 3

This composite part was designed to produce Phytase with linkers and cysteine modules on both termini, together with signal peptide on N terminus and His-Tag on C terminus. This structure is similar to INSERT 2, but without SAPs. In short, we can compare the influence of SAPs in circularization, and whether circularization make Phytase more competent or not.

INSERT 4

When INSERT 4 is translated, it produces Phytase with signal peptide on N terminus and His-Tag on C terminus. This is used for comparing how normal Phytase (this) and circularized Phytase (INSERT 2) are different, from the viewpoint of enzyme activity or resistance for tough environment.