Part:BBa_K5330021

This part along with BBa_K5330020 are the two components of a test for Mycobacterium avium subspecies paratuberculosis (MAP). This part is composed of a type 2A encapsulin (from MAP) with a linker to LgBiT. The LgBiT is half of a split luciferase. This acts as a reporter system for cage formation of Encapsulins linked to either LgBiT or SmBiT which when they come together in the presence of NanoGlo reagents, they produce light. With both SmBiT-Encap2A and LgBiT-Encap2A present in the test, light output is detected. When a blood sample of a cow infected with Johnes diseases is introduced into this solution, there should be a detected loss of light. 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.

Figure 1: Negative Test Diagram (no presence of MAP Encapsulin monomers).

Figure 2: Positive Test Diagram (presence of MAP Encapsulin monomers eg. a MAP infected blood sample).

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 588
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 754
  Illegal NgoMIV site found at 760
  
• 1000
  COMPATIBLE WITH RFC[1000]

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.

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

Figure 3: Type 2 Encapsulins (right) and Type 1 Encapsulins in their cage formations(1).

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(2) 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.

Figure 4: Type 2 Encapsulin attached to LgBiT.

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. This part was produced in SHuffle® T7 Competent E. coli due to their enhanced protein expression and folding ability. Due to the NEBuilder not taking up the LgBiT gene insert into the plasmid, we were unable to purify the protein in time to perform the assay with SmBiT-Encap to see if light would be produced.

Sources

Encapsulin Type 2A = UniProt ID I3NID5 · ENCP2_MYCPA

Linker = (2)

SmBiT = IGEM Registry BBa_K1761005

(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.

(2) (2)Choi, H.; Eom, S.; Kim, H.; Bae, Y.; Jung, H. S.; Kang, S. Load and Display: Engineering Encapsulin as a Modular Nanoplatform for Protein-Cargo Encapsulation and Protein-Ligand Decoration Using Split Intein and SpyTag/SpyCatcher. Biomacromolecules 2021, 22 (7), 3028–3039. https://doi.org/10.1021/acs.biomac.1c00481.