Difference between revisions of "Part:BBa K3815015"
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===Usage and Biology===
 
===Usage and Biology===
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===Improvement===
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[[File:improve 1 .png|300px|thumb|right|Fig.7 TmEncapsulin was purified more efficiently with Ni-NTA
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TmEncapsulin expressed E.coli was sonicated lysed with sonication. In the “Lysate” lane, the lysate was loaded. In the “Heat” lane, heat-treated supernatant was loaded. In the “Ni-NTA” lane, the protein was purified with Ni-NTA beads from the lysate, then loaded.]]
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[[File:Improve graph.png|300px|thumb|right|Fig.8 Ni-NTA beads purification got about 5 times concentrated TmEncapsulin
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SDS-PAGE that showed in Fig.7 was triplicated, then band intensities were quantified with ImageJ. Then each protein concentration of the solution was calculated.]]
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This part is our improved part. We improved “Encapsulin protein with HexaHistidine insert” (''<partinfo>BBa_K2686002</partinfo>'') to “SPYtag inserted Tm Encapsulin” (''<partinfo>BBa_K3185000</partinfo>''). We introduced two modifications to the existing part. First, we improved purification efficiency by adding 6x-His tag to C-terminus. Second, we enabled displaying different types of proteins on the surface of protein capsule by inserting SpyTag.
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<b>1st improve point: Improve purification efficiency by adding 6x-His tag to C-terminus</b>
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TmEncapsulin is a protein found from Thermotoga maritima. TmEncapsulin assembles sphere-like protein capsule as 60mer. This protein capsule could be applied to different situations, and ways to overexpress with E-coli and purify have been researched for a long time.
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Encapsulin on existing basic parts page promoted thermotolerance of native TmEncapsulin by introducing 6x-His tag between 43rd and 44th amino acid. After Expressing this by E.coli cell-free system, we can purify encapsulin by collecting supernatant after heat treatment and centrifugation.
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We added 6x-His tag to the C-terminus of an existing part. This is due to make them available to protein purify by using Ni-NTA beads. However, in a paper suggested, Ni-NTA beads cannot bind to 6x-His tag added in C-terminus because it doesn’t display enough to the surface of the protein capsule [4]. Here, we placed HAtag between TmEncapsulin and 6x-His tag in expectation of C-terminus to display enough on the surface of the capsule. We compared E.coli lysate before purification with a heat-treated product and Ni-NTA purified product with SDS-PAGE.
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Shown in the Fig.7 below, we succeeded in purifying Enacapuslin by using Ni-NTA beads. You can see that the product purified by Ni-NTA has a darker band. Also, we quantified band intensity, then calculated purified protein concentration. As shown in Fig.8, we were able to purify with five times as much concentration than the existing part by using Ni-NTA. This improvement means we can purify encapsulin more easily with high concentration.
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<b>2nd improve point: Protein display through SpyCatcher/SpyTag system</b>
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There are many possible usages of Encapsulin. One is protein displaying on the surface of the capsule. For example, iGEM EPFL 2018 which created the existing part also created Encapsulin with HexaHistidine insert and C-terminal OT1 (''<partinfo>BBa_K2686000</partinfo>'') which are antigens of “OT1”. This modification aims to display antigen to immune cells effectively.
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In order to make protein displaying easier, we inserted SpyTag in TmEncapsulin. SpyTag forms an isopeptide bond with SpyCatcher when they are mixed [3]. In previous research about TmEncap, it is showed peptides inserted after 138th amino acid in TmEncap can be exposed on the protein capsule as a loop [4]. Furthermore, Bae et al. showed when SpyTag is inserted at the same position, SpyCatcher/SpyTag also forms a bond between SpyCatcher and SpyTag inserted TmEncap (SpyTmEnc) [5]. This modification enables us to display different types of protein on SpyTmEnc through SpyCatcher. (See the Visual description of protein displaying)
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To demonstrate our SpyTmEnc can conjugate with SpyCatcher fused protein, we mixed SpyTmEncap with several kinds of protein. In lane 4 and 5, SpyTmEnc is loaded with or without SpyC. Only in lane 5, which is mixed with SpyC, the bigger band appeared. The molecular weight of each protein is SpyC: 15.37k, SpyTmEnc: 37.04k, so the conjugated band should be 52.41k. Therefore the bigger band looks objective conjugated protein. As a negative control, we also tested with an existing part: TmEncapsulin without SpyTag. Properly, TmEnc and SpyC did not show conjugated band as shown in lane 3. Also, as shown in lane 7 and 9, proteins fused with SpyCatcher are also conjugated to SpyTmEnc.
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This result shows we succeed in conjugating several kinds of proteins to Encapsulin. This means that the arbitrary type of protein which fused with SpyCatcher can be displayed on the protein capsule.
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Revision as of 12:48, 20 October 2021


AANDENYALGA. mutant SsrA degradation tag

This is an engineered derivative of wildtype ssrA tag from Escherichia coli, acquired by mutagenizing the WT tag with mixed primers. Compared to the WT, three C-terminal amino acids LAA are replaced to LGA. To quantify its efficiency of protein degradation, ... of E.coli overnight culture bearing a plasmid expressing GFP-ssrA tag fusion protein was measured by Qubit, and then compared to the WT and other mutants.


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]