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Team:Washington/Project
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| Target Vector | Secretion System | Display System |
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The target system is a biobrick compatible expression vector, that tags any favorite protein to be secreted, purified, and localized on the cell surface. To do this, we synthesized our “target construct” through gene synthesis to have a nano-tag, two histidine tags, two TEV protease sights, an Nhe1 cut site, and prtB (need biobrick name) and terminator BBa_B0015(all need referenecs). The Nhe1 sticky ends are compatible with Xba1 and Spe1 sticky ends, allowing for the insertion of biobricked genes into the target construct. The nano-tag is a short sequence that allows the target construct to non-covalently bond to the surface protein streptavadin, in a highly characterized interaction (reference). The Harvard part [http://partsregistry.org/wiki/index.php/Part:BBa_J36848 J36848] from 2006, a monomeric form of streptavadin theoretically allows the protein to linger on the cell till a convenient time for purification, though we had difficulty getting functional protein (please reference display team). TEV protease sights flank the Nhe1 sight allowing the desired protein to be excised from the target construct if desired during or after purification. Histidine tags are on the outsides of the TEV sights allowing the target construct with a favorite protein to be purified via the traditional purification methods, or to remove fragments of the target construct after TEV cutting, leaving highly purified protein behind.
