Difference between revisions of "Part:BBa K3185004"

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As our project involves connecting different proteins, we decided to use the SpyTag/SpyCatcher system and have inserted the SpyCatcher at the N-terminal of the BaCBM2. When this part connected with SpyTag inserted Tm Encapsulin, the compound does complete work as flocculant(りんく).
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As our project involves connecting different proteins, we decided to use the SpyTag/SpyCatcher system and have inserted the SpyCatcher at the N-terminal of the BaCBM2. When this part connected with SpyTag inserted Tm Encapsulin(りんく), the compound does complete work as flocculant.
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This part has three tags or binding sites;6xHis-tag,MYC-tag and TEV-binding site.6xHis-tag is inserted at the N-terminal of the SpyCatcher and used for affinity purification of this part.MYC-tag is also inserted between SpyCatcher and BaCBM2 in order to be detected with antiboby in Western brotting.TEV-binding site is inserted in the original article[2], and we left it as it was so as not to decrease the function, although the site is not used in our experiment.
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This part has three tags or binding sites;6xHis-tag,MYC-tag and TEV-binding site.6xHis-tag is inserted at the N-terminal of the SpyCatcher and used for affinity purification of this part.MYC-tag is also inserted between SpyCatcher and BaCBM2 in order to be detected with antiboby in Western brotting.TEV-binding site is inserted in the original article[2], and we left it as it was so as not to change the function, although the site is not used in our experiment.
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We insert this part in pET-11a plasmid between BamHI and NdeI restriction sites.
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We lay this part on pET-11a plasmid between BamHI and NdeI restriction sites.
  
  

Revision as of 13:14, 8 October 2019


SPYCatcher -> BaCBM2

Usage and Biology

BaCBM2 is a Carbonhydrate-binding Module family 2(CBM2) derived from Bacillus anthracis.CBMs are protein domains mainly found in carbohydrate-active enzymes.According to an article[1], BaCBM2 is proved to have the highest binding ability to PET film, so we introduce this protain to the part as PET-binding protain.

As our project involves connecting different proteins, we decided to use the SpyTag/SpyCatcher system and have inserted the SpyCatcher at the N-terminal of the BaCBM2. When this part connected with SpyTag inserted Tm Encapsulin(りんく), the compound does complete work as flocculant.

This part has three tags or binding sites;6xHis-tag,MYC-tag and TEV-binding site.6xHis-tag is inserted at the N-terminal of the SpyCatcher and used for affinity purification of this part.MYC-tag is also inserted between SpyCatcher and BaCBM2 in order to be detected with antiboby in Western brotting.TEV-binding site is inserted in the original article[2], and we left it as it was so as not to change the function, although the site is not used in our experiment.

We lay this part on pET-11a plasmid between BamHI and NdeI restriction sites.


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]

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