Difference between revisions of "Part:BBa K4275011"
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[[File:GreatBay SCIE--3D PETase-5-t.png|800px]]
 
[[File:GreatBay SCIE--3D PETase-5-t.png|800px]]
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<p align="center"><b>Figure 1</b> The 3D structure of the protein predicted by Alphafold2. </p>
  
 
===Usage and Biology===
 
===Usage and Biology===
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The artificially-designed PETase5 - Dockerin I fusion protein could be tightly-anchored onto the CipA scaffoldin via the high-affinity Doc I: Coh I noncovalent interaction. The CipA primary scaffoldin is then tightly-anchored onto the secondary scaffoldin - OlpB, which is either anchored onto the cell surface of K.marxianus via ScGPI, or binds to E. coli's Cell-surface Nanobody3(Nb3). It is believed that the immobilization of the two enzymes (PETase5-t and MHETase-t) could increase their proximity and further enhance their synergy, whilst the affinity of carbohydrate-binding module 3 (CBM3) on the CipA scaffoldin towards PET fiber could further increase the catalytic efficiency of this degradation complex.
 
  
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The artificially-designed PETase5-Dockerin I fusion protein could be tightly-anchored onto the CipA scaffoldin via the high-affinity Doc I: Coh I noncovalent interaction. The CipA primary scaffoldin is then tightly-anchored onto the secondary scaffoldin - OlpB, which is either anchored onto the cell surface of <i>K.marxianus</i> via ScGPI, or binds to <i>E.coli</i>'s Cell-surface Nanobody3(Nb3)(BBa_K4275026). It is believed that the immobilization of the two enzymes (PETase5-dockerin and MHETase-t(BBa_K4275010)) could increase their proximity and further enhance their synergy, whilst the affinity of carbohydrate-binding module 3 (CBM3) on the CipA scaffoldin towards PET fiber could further increase the catalytic efficiency of this degradation complex.
  
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===Design consideration===
 
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1. A 10 aa long GS linker (GGGGS)2 is appended following the Ser273 residue at the C' terminal of the original sequence.
 
  
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2. The 79 amino acid long type-I dockerin domain is fused at the terminal of the GS linker, followed by a TEV site and a 6xhis affinity purification tag (HHHHHH).
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===Sequence and Features===
  
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<span class='h3bb'>Sequence and Features</span>
 
 
<partinfo>BBa_K4275011 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4275011 SequenceAndFeatures</partinfo>
  

Revision as of 00:01, 12 October 2022


PETase5-dockerin

PETase5-dockerin is an improved version of Super-5-mut PET hydrolase from the iGEM team TJUSLS_China (Part: BBa_K3715005). This high-efficiency, thermostable, durable super mutant consists of 11 mutation sites compared to the wild-type: S214H, I168R, W159H, S188Q, R280A, A180I, G165A, Q119Y, L117F, T140D, S121E. The improvement is implemented by fusing the original sequence design with a dockerin I domain at the C' terminal to allow its high-affinity anchorage onto the CipA scaffoldin and the rest of the polyester degradation complex. The catalytic domain of PETase5-t and the dockerin domain are interspaced with a medium-lengthed flexible GS linker (10 aa long) to avoid steric inhibitions.

GreatBay SCIE--3D PETase-5-t.png

Figure 1 The 3D structure of the protein predicted by Alphafold2.

Usage and Biology

The artificially-designed PETase5-Dockerin I fusion protein could be tightly-anchored onto the CipA scaffoldin via the high-affinity Doc I: Coh I noncovalent interaction. The CipA primary scaffoldin is then tightly-anchored onto the secondary scaffoldin - OlpB, which is either anchored onto the cell surface of K.marxianus via ScGPI, or binds to E.coli's Cell-surface Nanobody3(Nb3)(BBa_K4275026). It is believed that the immobilization of the two enzymes (PETase5-dockerin and MHETase-t(BBa_K4275010)) could increase their proximity and further enhance their synergy, whilst the affinity of carbohydrate-binding module 3 (CBM3) on the CipA scaffoldin towards PET fiber could further increase the catalytic efficiency of this degradation complex.


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
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 88
    Illegal NgoMIV site found at 142
    Illegal NgoMIV site found at 169
  • 1000
    COMPATIBLE WITH RFC[1000]