Difference between revisions of "Part:BBa K5330019"

Revision as of 23:17, 25 September 2024

SmBiT-Encapsulin2A

This part is a subtype of encapsulin specific to Mycobacterium avium subspecies paratuberculosis (MAP) encoded by gene MAP2121C. Encapsulins are cage forming proteins that are used for metabolic channelling/substrate protection in lower organisms. Encapsulin monomers readily form multimeric cages under variable conditions resulting in labile exchange of monomers to make varying cage sizes. Type 2A encapsulins are less well studied that type 1. The differentiation usually arises from the species of organism and grouped along with what metabolic pathways they are linked to. In the case of Type 2A, they are linked to sulphur metabolism and are found on same chromosome as a group II cysteine desulfurase, likely on the same operon. We chose to purify this protein because we wanted to characterise it but also as we needed a positive control for our bioluminescent assay. In conjugation with part BBa_K5330020 and BBa_K5330021, this will theoretically produce a loss of light when mixed with NanoGlo. This part will be used to emulate a blood sample containing MAP and thus MAP specific proteins. This is because Encapsulin monomers from MAP will rearrange to incorporate our engineered monomers (SmBiT-Encap2A and LgBiT-Encap2A) resulting in distance being introduced between halves of the split luciferase and either less or no light produced at all. Type 1 Encapsulins have been submitted to the registry before BBa_K192000 with use in encapsulating enzymes for metabolic channeling.

Sequence and Features

Design considerations

In the design of this fusion protein there were a few things we had to consider. Type 2A encapsulins are much less well characterised as Type 1 encapsulins. This meant differentiating between the two in terms of size of cages, overall charge and pore size and location. Then we decided how long we wanted our linker; did we want more flexibility, or did we want a bit more control over how far we can reach with the two split luciferase parts. We found a paper (Choi et al., 2021) where they attached NanoLuc to type 1 encapsulins to detect cage formation. From there, we copied their linker design (for which they did not describe the rational for) to attach our split luciferase parts and our type 2A encapsulin monomers. We have since encountered problems in our protein purification leading us to believe that our His tag should be on the C terminus (rather that the N terminus it currently is on.) This hinderance means that it is highly likely that the protein purification thus far will have produced monomers/cages that are not correctly formed and that could possibly be aggregates. This part is currently produced in a PC2 lab due to the production being from a GMO. In future, (see New Zealand laws about whether this protein could ever be exported from the lab). This part was produced in (talk to Jovarn about cell line/strain).

Experiments run to test if the part worked involved mixing it with the NanoGlo and BBa_K5330021 and trying to detect light. (see further experiments from objective 3).

Source

Encapsulin Type 2A = UniProt ID I3NID5 · ENCP2_MYCPA

Linker = Hyukjun Choi, Soomin Eom, Han-ul Kim, Yoonji Bae, Hyun Suk Jung, and Sebyung Kang Biomacromolecules 2021 22 (7), 3028-3039. DOI: 10.1021/acs.biomac.1c00481

LgBiT = IGEM Registry BBa_K1761005