Difference between revisions of "Part:BBa K3185010"
 
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<partinfo>BBa_K3185010 short</partinfo>
 
<partinfo>BBa_K3185010 short</partinfo>
 
==Usage and Biology==
 
==Usage and Biology==
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Engineered PETase is a protein from Ideonella sakaiensis. The paper (Characterization and engineering of a plastic-degrading aromatic polyesterase) tries to improve the binding activity and the degradation activity of PET.  
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Engineered PETase is a protein from <i>Ideonella sakaiensis</i>. In a paper, the binding activity and the degradation activity of the PETase enzyme were both improved by introducing mutations [1].
 
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This time, we used engineered PETase which shows a higher binding affinity to PET than PETase in order to compare them. We put SpyCatcher on N-terminus of PETase because we used SpyTag/SpyCatcher system to bind it to other parts.  
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We used engineered PETase which shows a higher binding affinity to PET than PETase in order to compare them. We put SpyCatcher(''<partinfo>BBa_K1159200</Partinfo>'') on N-terminus of PETase because we used SpyCatcher/SpyTag system to bind it to other parts.  
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It has three tags and a cleavage site. First is 6×His-tag inserted in the N-terminus of SpyCather for protein purification. Second is MYC-tag inserted between SpyCatcher and CBM (Cellose binding module ??more detail??) 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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It has three tags and a cleavage site. First is 6×-His tag inserted in the N-terminus of SpyCather for protein purification. Second is MYC-tag inserted between SpyCatcher and PETase to detect it by using the antibody. Third is a TEV protease site because, in the paper, it was used for protein purification [2]. However, we didn’t use it in our experiment.  
 
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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 EngiPETase by using SDS-PAGE.
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We put it between BamHI site and Ndel site on pET11-a. The expression plasmids were introduced into BL21(DE3) and expressed by T7 promoter/ T7 RNAP system. Ni-NTA agarose was used for the purification.
  
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K3185010 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3185010 SequenceAndFeatures</partinfo>
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==Purification==
 
==Purification==
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[[File:engineered PETase.png|300px|thumb|right|Fig.1 SDS-PAGE of imidazole elutes, CBB stained]]
 
<h3><font size="4.5">Expression</font> </h3>
 
<h3><font size="4.5">Expression</font> </h3>
 
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<li>Protein was expressed in 0.1mM IPTG for 2hours.
 
<li>Protein was expressed in 0.1mM IPTG for 2hours.
 
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</ul>
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<h3><font size="4.5">SDS-PAGE</font></h3>
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<h3><font size="4.5">Purification </font></h3>
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1. <I>E.coli</i> which expressed this part were lysed with sonification.<br>
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2. Proteins are purified from lysate with Ni-NTA agarose(QIAGEN).<br>
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3. Imidazole eluates were visualized and confirmed by SDS-PAGE followed by CBB staining.<br>
 
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This purification method failed. As shown in Fig.1, we didn't see any band in the specific molecular weight of the protein.
 
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==Result==
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We couldn't get any result in our experiment because we failed the protein purification.
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==References==
 
==References==
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1 Austin, H.P., Allen, M.D., Donohoe, B.S., Rorrer, N.A., Kearns, F.L., Silveira, R.L., Pollard, B.C., Dominick, G., Duman, R., Omari, K. El, et al. (2018).
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<br>
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Characterization and engineering of a plastic-degrading aromatic polyesterase.
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<br>
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<i>Proc. Natl. Acad. Sci. U. S. A. </i>115, E4350–E4357.
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2 Veggiani, G., Nakamura, T., Brenner, M.D., Gayet, R. V., Yan, J., Robinson, C. V., and Howarth, M. (2016).
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Programmable polyproteams built using twin peptide superglues.
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<i>Proc. Natl. Acad. Sci. U. S. A. </i>113, 1202–1207.
 
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===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K3185010 parameters</partinfo>
 
<partinfo>BBa_K3185010 parameters</partinfo>
 
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Latest revision as of 03:14, 22 October 2019


SPYCatcher -> engineered PETase

Usage and Biology

Engineered PETase is a protein from Ideonella sakaiensis. In a paper, the binding activity and the degradation activity of the PETase enzyme were both improved by introducing mutations [1].

We used engineered PETase which shows a higher binding affinity to PET than PETase in order to compare them. We put SpyCatcher(BBa_K1159200) on N-terminus of PETase because we used SpyCatcher/SpyTag system to bind it to other parts. It has three tags and a cleavage site. First is 6×-His tag inserted in the N-terminus of SpyCather for protein purification. Second is MYC-tag inserted between SpyCatcher and PETase to detect it by using the antibody. Third is a TEV protease site because, in the paper, it was used for protein purification [2]. However, we didn’t use it in our experiment.

We put it between BamHI site and Ndel site on pET11-a. The expression plasmids were introduced into BL21(DE3) and expressed by T7 promoter/ T7 RNAP system. Ni-NTA agarose was used for the purification.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 751
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 1318
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1074
  • 1000
    COMPATIBLE WITH RFC[1000]

Purification

Fig.1 SDS-PAGE of imidazole elutes, CBB stained

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.

Purification

1. E.coli which expressed this part were lysed with sonification.
2. Proteins are purified from lysate with Ni-NTA agarose(QIAGEN).
3. Imidazole eluates were visualized and confirmed by SDS-PAGE followed by CBB staining.

This purification method failed. As shown in Fig.1, we didn't see any band in the specific molecular weight of the protein.

Result

We couldn't get any result in our experiment because we failed the protein purification.

References

1 Austin, H.P., Allen, M.D., Donohoe, B.S., Rorrer, N.A., Kearns, F.L., Silveira, R.L., Pollard, B.C., Dominick, G., Duman, R., Omari, K. El, et al. (2018).
Characterization and engineering of a plastic-degrading aromatic polyesterase.
Proc. Natl. Acad. Sci. U. S. A. 115, E4350–E4357.

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.