All the parts we made.
We developed a total of twelve new basic parts, eight of which were Reb proteins with linkers engineered onto an end. These new basic Reb proteins were then assembled into eight new corresponding constructs supporting our conjugation strategies.
| Part | Name | Purpose |
|---|---|---|
| BBa_K5121005 | rebC | A small component of the R body protein. |
| BBa_K5121006 | rebD | A minor component of the R body protein, with an unknown role. |
| BBa_K5121007 | T7 Promoter from Reb1 | The promoter we used to express Reb proteins. |
| BBa_K5121012 | GGG-mNeon | A fluorescent reporter for testing Sortase A conjugation. |
| BBa_K5121013 | rebA N-terminal CGGGGS | rebA, a major structural subunit of the protein, with a flexible linker preparing it for conjugation with maleimides on its N terminus. |
| BBa_K5121014 | rebA C-terminal SGGGGC | rebA, a major structural subunit of the protein, with a flexible linker preparing it for conjugation with maleimides on its C terminus. |
| BBa_K5121015 | rebB N-terminal CGGGGS | rebB, a major structural subunit of the protein, with a flexible linker preparing it for conjugation with maleimides on its N terminus. |
| BBa_K5121016 | rebB C-terminal SGGGGC | rebB, a major structural subunit of the protein, with a flexible linker preparing it for conjugation with maleimides on its C terminus. |
| BBa_K5121017 | rebA N-terminal TAG | rebA, a major structural subunit of the protein, modified to have an amber stop codon (TAG) on its N terminus. This amber stop codon can be used to attach non-canonical amino acids, which allows for a variety of conjugation methods. |
| BBa_K5121018 | rebB N-terminal TAG | rebB, a major structural subunit of R bodies, modified to have an amber stop codon (TAG) on its N terminus. This amber stop codon can be used to attach non-canonical amino acids, which allows for a variety of conjugation methods. |
| BBa_K5121019 | rebA C-teriminal LPETG | rebA, a major structural subunit of R bodies, with an LPETG linker on its C terminus, which the enzyme Sortase A recognises and cleaves, conjugating it with another protein possessing a GGG on one of its ends. |
| BBa_K5121020 | rebB C-terminal LPETG | rebB, a major structural subunit of R bodies, with an LPETG linker on its C terminus, which the enzyme Sortase A recognises and cleaves, conjugating it with another protein possessing a GGG on one of its ends. |
| Part | Name | Purpose |
|---|---|---|
| BBa_K5121021 | Reb1 with N-terminal CGGGGS on rebA | The full R body protein, with a linker on rebA's N terminus for cysteine-maleimide conjugation. |
| BBa_K5121022 | Reb1 with C-terminal SGGGGC on rebA | The full R body protein, with a linker on rebA's C terminus for cysteine-maleimide conjugation. |
| BBa_K5121023 | Reb1 with N-terminal CGGGGS on rebB | The full R body protein, with a linker on rebB's N terminus for cysteine-maleimide conjugation. |
| BBa_K5121024 | Reb1 with C-terminal SGGGGC on rebB | The full R body protein, with a linker for rebB's C terminus for cysteine-maleimide conjugation. |
| BBa_K5121025 | Reb1 with N-terminal TAG on rebA | The full R body protein, with a linker for rebA's N terminus for amber stop codon suppression for incorporating conjugation-ready non-canonical amino acids into R bodies. |
| BBa_K5121026 | Reb1 with N-terminal TAG on rebB | The full R body protein, with a linker for rebB's N terminus for amber stop codon suppression for incorporating conjugation-ready non-canonical amino acids into R bodies. |
| BBa_K5121027 | Reb1 with C-terminal LPETG on rebA | The full R body protein, with a linker for rebA's C terminus for the enzyme Sortase A to cleave off and specifically conjugate proteins to R bodies. |
| BBa_K5121028 | Reb1 with C-terminal LPETG on rebB | The full R body protein, with a linker for rebB's C terminus for the enzyme Sortase A to cleave off and specifically conjugate proteins to R bodies. |