Team:Queens Canada/Basic Part




CsgA-SpyTag Protein



The CsgA-SpyTag Fusion Protein is one half of a split protein system. When SpyTag finds SpyCatcher in solution, they form a covalent bond, joining whatever is fused onto each part of the split protein system. SpyTag is fused onto CsgA (an E. coli biofilm amyloid protein) so that large proteins can be added to the biofilm that are larger than the export limitation of around 60 amino acids.

Golden Gate Assembly



Allows golden gate assembly onto an E. coli biofilm amyloid protein (CsgA) so that a variety of fusion proteins can easily be made. The efficient and seamless assembly of DNA fragments, commonly referred to as Golden Gate assembly, has its origins in 1996, when for the first time it was shown that multiple inserts could be assembled into a vector backbone using only the sequential or simultaneous activities of a single type IIS restriction enzyme and T4 DNA ligase. Golden Gate Assembly and its derivative methods exploit the ability of Type IIS restriction endonucleases (REases) to cleave DNA outside of the recognition sequence. The inserts and cloning vectors are designed to place the Type IIS recognition site distal to the cleavage site, such that the Type IIS REase can remove the recognition sequence from the assembly. The net result is the ordered and seamless assembly of DNA fragments in one reaction.

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