Difference between revisions of "Part:BBa K3185004"

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<partinfo>BBa_K3185004 short</partinfo>
 
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==Usage and Biology==
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==Usage and Biology=
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BaCBM2 is a Carbonhydrate-binding Module family 2(CBM2) derived from <i>Bacillus anthracis</i>.  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.
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BaCBM2 is a Carbohydrate-Binding Module (CBM) from Bacillus anthracis.  CBM often found in Carbohydrate related enzymes.  It can bind to not only highly crystallized cellulose but also PET because it has a binding site formed by aromatic amino acids. (Boraston et al. 2004) (Carbohydrate-binding modules: fine-tuning polysaccharide recognition) In this paper (Interaction of carbohydrate-binding modules with poly (ethylene terephthalate)), they research binding affinity of some kinds of CBM and PET. As a result, it is found that BaCBM2 has the most strong binding affinity to PET.
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We used BaCBM2 as PET binding domain. We put SpyCatcher on N-terminus of BaCBM2 because we used SpyTag/SpyCatcher system to bind it to other parts. Also, this has three tag and cleavage sites. First is 6×His-tag inserted in the N-terminus of SpyC for protein purification. Second is MYC-tag inserted between SpyC and CBM to detect it by using the antibody. Third is a TEV protease site because, in the paper, it was used for protein purification. (Programmable polyproteins built using twin peptide superglues) However, we didn’t use it in our experiment.
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We put it between BamHI site and Ndel site on pET11-a. We used BL21 (DE3) for gene expression. We used Ni-NTA Agarose for purification. After that, we confirmed molecular weight of BaCBM2 by using SDS-PAGE.
 
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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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==Purification==
 
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<h3><font size="4.5"> 中見出し</font> </h3>
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<h3><font size="4.5"> 中見出し</font></h3>
 
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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 lay this part on pET-11a plasmid between BamHI and NdeI restriction sites, and using the T7 promoter to express the resulting protein in BL21(DE3).
 
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<span class='h3bb'>Sequence and Features</span>
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=Sequence and Features=
 
<partinfo>BBa_K3185004 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3185004 SequenceAndFeatures</partinfo>
  

Revision as of 13:20, 17 October 2019


SPYCatcher -> BaCBM2

=Usage and Biology

BaCBM2 is a Carbohydrate-Binding Module (CBM) from Bacillus anthracis. CBM often found in Carbohydrate related enzymes. It can bind to not only highly crystallized cellulose but also PET because it has a binding site formed by aromatic amino acids. (Boraston et al. 2004) (Carbohydrate-binding modules: fine-tuning polysaccharide recognition) In this paper (Interaction of carbohydrate-binding modules with poly (ethylene terephthalate)), they research binding affinity of some kinds of CBM and PET. As a result, it is found that BaCBM2 has the most strong binding affinity to PET. 

We used BaCBM2 as PET binding domain. We put SpyCatcher on N-terminus of BaCBM2 because we used SpyTag/SpyCatcher system to bind it to other parts. Also, this has three tag and cleavage sites. First is 6×His-tag inserted in the N-terminus of SpyC for protein purification. Second is MYC-tag inserted between SpyC and CBM to detect it by using the antibody. Third is a TEV protease site because, in the paper, it was used for protein purification. (Programmable polyproteins built using twin peptide superglues) However, we didn’t use it in our experiment.

We put it between BamHI site and Ndel site on pET11-a. We used BL21 (DE3) for gene expression. We used Ni-NTA Agarose for purification. After that, we confirmed molecular weight of BaCBM2 by using SDS-PAGE.

Purification


中見出し

中見出し




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