Difference between revisions of "Part:BBa K3185000"

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==Result==
 
==Result==
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[[File:191001 SPYt-SPYC 2.png|300px|thumb|left|Fig. 10 Isopeptide bond formation between Plastic binding proteins and Encapsulin.
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[[File:peak191015.png|300px|thumb|left|Fig. 1 TmEncapsulin polymer appears as the peak.
 
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3µL of SpyCatcher-Plastic-binding protein (SpyC-PBP) solution and 3µL of SpyTag inserted TmEncapsulin (SpyTmEnc) solution was mixed, then placed for 16h at room temperature.  Then 6µL of 2x SDS sample buffer was added.  10µL of each sample was loaded.  SDS-PAGE for 30min in 200V.  The gel was CBB stained.]]
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TmEncapsulin expressed E. coli lysate and purified protein solution was loaded on 10%-60% sucrose linear gradient / 20 mM Tris 7.5, 50 mM NaCl, then centrifuged in 100,000g for 18 hours at 4℃ with SW41(Beckman).  The solution was fractionated on a 96-well plate with BioComp.  At the same time, 260nm absorption was measured. ]]
 
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[[File:peak191015.png|300px|thumb|left|Fig. 8 TmEncapsulin polymer appears as the peak.
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[[File:191001 SPYt-SPYC 2.png|300px|thumb|left|Fig. 2 Isopeptide bond formation between Plastic binding proteins and Encapsulin.
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TmEncapsulin expressed E. coli lysate and purified protein solution was loaded on 10%-60% sucrose linear gradient / 20 mM Tris 7.5, 50 mM NaCl, then centrifuged in 100,000g for 18 hours at 4℃ with SW41(Beckman).  The solution was fractionated on a 96-well plate with BioComp.  At the same time, 260nm absorption was measured. ]]
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3µL of SpyCatcher-Plastic-binding protein (SpyC-PBP) solution and 3µL of SpyTag inserted TmEncapsulin (SpyTmEnc) solution was mixed, then placed for 16h at room temperature.  Then 6µL of 2x SDS sample buffer was added.  10µL of each sample was loaded.  SDS-PAGE for 30min in 200V.  The gel was CBB stained.]]
 
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[[File:SpyC-T timeD.png|300px|thumb|left|Fig. 12 Quantification of conjugated band
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[[File:SpyC-T timeD.png|300px|thumb|left|Fig. 3 Quantification of conjugated band
 
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Conjugated bands’ intensity was quantified with ImageJ.  Orange dots show averages value of three experiments.  Blacklines show standard deviations.  The time point 60min was deleted because it includes negative value.
 
Conjugated bands’ intensity was quantified with ImageJ.  Orange dots show averages value of three experiments.  Blacklines show standard deviations.  The time point 60min was deleted because it includes negative value.

Revision as of 10:32, 19 October 2019


SPYtag inserted Tm Encapsulin

Usage and Biology

TmEncapsulin is a protein found from Thermotoga maritima. A paper says that it consists of 60 monomers and forms capsule, Virus-like particle(VLP)[1]. iGEM also treats it as a useful part (BBa_K192000).

We used TmEncapsulin as a biological polymer. We put Spytag inside TmEncapsulin because we used SpyTag/SpyCatcher system to bind it to other parts(SpyCatcher:BBa_K1159200, SpyTag:BBa_K1159201)[2]. Also, this has three tag and cleavage sites. First is 6×His-tag inserted in the C-terminus of TmEncapsulin for protein purification. Second is HA-tag inserted between TmEncapsulin and 6x-His-tag to detect it by using antibodies. Third is a 6x-His tag inserted between the C-terminus of Encapsulin and 6x-His-tag because, in a paper, it is said that 6x-His-tag inserted in the C-terminus of Encapsulin is not presented on the surface of Encapsulin well, so it can’t bind to Ni-NTA Agarose beads. In the same paper, it is also said that heat-resistance is improved when inserting 6x-His-tag and linker between #43 and #44 amino acids of native encapsulin, so we designed it like that[3].

We put it between the BamHI site and the Ndel site on pET11-a. We used BL21 (DE3) for gene expression. We used the Ni-NTA Agarose for purification. After that, we confirmed the molecular weight of SpyCatcher inserted TmEncapusulin by using SDS-PAGE.

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 77
    Illegal BglII site found at 597
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 426
    Illegal SapI.rc site found at 457

Purification


Expression

  • Cells were grown in 200ml LB media (100μg/ml Ampicillin) at 37oC shaking at 140 rpm to an OD600 of 0.5, verifying via a spectrophotometer.
  • Protein was expressed in 0.1mM IPTG for 2hours.

SDS-PAGE

alt text



Result

Fig. 1 TmEncapsulin polymer appears as the peak.
TmEncapsulin expressed E. coli lysate and purified protein solution was loaded on 10%-60% sucrose linear gradient / 20 mM Tris 7.5, 50 mM NaCl, then centrifuged in 100,000g for 18 hours at 4℃ with SW41(Beckman). The solution was fractionated on a 96-well plate with BioComp. At the same time, 260nm absorption was measured.



Fig. 2 Isopeptide bond formation between Plastic binding proteins and Encapsulin.
3µL of SpyCatcher-Plastic-binding protein (SpyC-PBP) solution and 3µL of SpyTag inserted TmEncapsulin (SpyTmEnc) solution was mixed, then placed for 16h at room temperature. Then 6µL of 2x SDS sample buffer was added. 10µL of each sample was loaded. SDS-PAGE for 30min in 200V. The gel was CBB stained.



Fig. 3 Quantification of conjugated band
Conjugated bands’ intensity was quantified with ImageJ. Orange dots show averages value of three experiments. Blacklines show standard deviations. The time point 60min was deleted because it includes negative value.

References

1 Putri, R.M., Allende-Ballestero, C., Luque, D., Klem, R., Rousou, K.A., Liu, A., Traulsen, C.H.H., Rurup, W.F., Koay, M.S.T., Castón, J.R., et al. (2017).
Structural Characterization of Native and Modified Encapsulins as Nanoplatforms for in Vitro Catalysis and Cellular Uptake.
ACS Nano 11, 12796–12804.

2 Veggiani, G., Nakamura, T., Brenner, M.D., Gayet, R. V., Yan, J., Robinson, C. V., and Howarth, M. (2016).
Programmable polyproteams built using twin peptide superglues.
Proc. Natl. Acad. Sci. U. S. A. 113, 1202–1207.

3 Moon, H., Lee, J., Min, J., and Kang, S. (2014).
Developing genetically engineered encapsulin protein cage nanoparticles as a targeted delivery nanoplatform.
Biomacromolecules 15, 3794–3801.