Difference between revisions of "Part:BBa K5330019"
 
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<partinfo>BBa_K5330020 short</partinfo>
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<partinfo>BBa_K5330019 short</partinfo>
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This part is a subtype of encapsulin specific to <I>Mycobacterium avium subspecies paratuberculosis </I> (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.
  
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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.
 
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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.
 
Type 1 Encapsulins have been submitted to the registry before BBa_K192000 with use in encapsulating enzymes for metabolic channeling.
  
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== Design considerations ==
 
== Design considerations ==
  
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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.
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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.  
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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).
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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).
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<center><img src = "https://static.igem.wiki/teams/5330/registry-parts/screenshot-2024-09-26-at-12-03-43-pm.png"></center>
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''Figure 1: Overlay of Type 1 and Type 2 Encapsulin monomers to show homology and heterology in structure(1).''
  
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== Source ==
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<center><img src = "https://static.igem.wiki/teams/5330/registry-parts/screenshot-2024-09-26-at-12-03-56-pm.png"></center>
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</html>
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''Figure 2: Type 2 Encapsulins (left) and Type 1 Encapsulins (right) in their cage formations(1).''
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We have since encountered problems in our protein purification using His tag purification on immobilised metal affinity chromatography. We think our His tag should have been on the C terminus (rather that the N terminus it currently is on.) This has lead to a less efficient purification with a lot of protein lost in the follow-through. For information of experimental data collected from this part, see the design page.
  
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Encapsulin Type 2A = UniProt ID I3NID5 · ENCP2_MYCPA
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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. As of the registry freeze, we are yet to achieve proof of concept due to complications in LgBiT purification.
  
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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
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== Usage in the UCNZ IGEM 2024 Project ==
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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.
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<html>
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<center><img src = "https://static.igem.wiki/teams/5330/registry-parts/screenshot-2024-09-26-at-1-14-41-pm.png"></center>
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</html>
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''Figure 1: Negative Test Diagram (no presence of MAP Encapsulin monomers).''
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<center><img src = "https://static.igem.wiki/teams/5330/registry-parts/screenshot-2024-09-26-at-1-22-28-pm.png"></center>
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''Figure 2: Positive Test Diagram (presence of MAP Encapsulin monomers eg. a MAP infected blood sample).''
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== Sources ==
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Encapsulin Type 2A = UniProt ID I3NID5 · ENCP2_MYCPA
  
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LgBiT = IGEM Registry BBa_K1761005
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(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.

Latest revision as of 22:19, 1 October 2024

Encapsulin 2A

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.

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 (left) and Type 1 Encapsulins (right) in their cage formations(1).

We have since encountered problems in our protein purification using His tag purification on immobilised metal affinity chromatography. We think our His tag should have been on the C terminus (rather that the N terminus it currently is on.) This has lead to a less efficient purification with a lot of protein lost in the follow-through. For information of experimental data collected from this part, see the design page.

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. As of the registry freeze, we are yet to achieve proof of concept due to complications in LgBiT purification.

Usage in the UCNZ IGEM 2024 Project

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.

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).

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.