Difference between revisions of "Part:BBa K5330021"

Latest revision as of 22:15, 1 October 2024

LgBiTEncapsulin2A

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 a LgBiT with a total molecular weight of 54kDa. 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 so 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 (see Figure 1).

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

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 (see Figure 2). This occurs due to the lability in exchange of monomers in encapsulin cages allowing interconversion of MAP encapsulin and our engineered ones.

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.

Laboratory

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.

Sources

Encapsulin Type 2A = UniProt ID I3NID5 · ENCP2_MYCPA

Linker = (2)

LgBiT = 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.

@@ Line 2: / Line 2: @@
 <partinfo>BBa_K5330021 short</partinfo>
-This part along with BBa_K5330020 are the two components of a test for <I> Mycobacterium avium subspecies paratuberculosis </I> (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 encapsulin. This works by having the engineered monomers linked to either LgBiT or SmBiT so that 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.
+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 a LgBiT with a total molecular weight of 54kDa. 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 so 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 (see Figure 1).
 <html>
@@ Line 8: / Line 8: @@
 </html>
 ''Figure 1: Negative Test Diagram (no presence of MAP Encapsulin monomers).''
+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 (see Figure 2). This occurs due to the lability in exchange of monomers in encapsulin cages allowing interconversion of MAP encapsulin and our engineered ones.
 <html>
@@ Line 21: / Line 23: @@
 == Design considerations ==
-In the design of this fusion protein there were a few things we had to consider.
+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.
-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.
 <html>
@@ Line 44: / Line 44: @@
 == Laboratory ==
-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.
+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.
-Another problem was that the NEBuilder did not take up the LgBiT gene insert into the plasmid. This meant that we were unable to purify the protein in time to perform the assay with SmBiT-Encap to see if light would be produced.
-We tried to express this part in a PC2 lab due to the production being from a GMO. We chose to express in SHuffle T7 Competent E. coli due to their enhanced protein expression and folding ability.
+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.
 == Sources ==
@@ Line 56: / Line 54: @@
 Linker = (2)
-SmBiT = IGEM Registry BBa_K1761005
+LgBiT = 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.