Part:BBa_K5330019

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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Design considerations

In the protein purification 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, pore size, localisation and cage forming conditions.

Figure 1: Overlay of Type 1 and Type 2 Encapsulin monomers to show homology and heterology in structure(1).

Figure 2: Type 2 Encapsulins (right) and Type 1 Encapsulins in their cage formations(1).

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). – shuffle cells for expression (because what we had, competence and availability), top 10 – high plasmid production

Experiments run to test if the cages are forming, and the protein is purified involve (see future experiments).

AUC – what will this show? Static Light Scattering (SLS) – what will this show?

Sources

Encapsulin Type 2A = UniProt ID I3NID5 · ENCP2_MYCPA

(1) Nichols, R. J.; LaFrance, B.; Phillips, N. R.; Radford, D. R.; Oltrogge, L. M.; Valentin-Alvarado, L. E.; Bischoff, A. J.; Nogales, E.; Savage, D. F. Discovery and Characterization of a Novel Family of Prokaryotic Nanocompartments Involved in Sulfur Metabolism. eLife 2021, 10, e59288. https://doi.org/10.7554/eLife.59288.