Difference between revisions of "Part:BBa K4275008"
 
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<partinfo>BBa_K4275008 short</partinfo>
 
<partinfo>BBa_K4275008 short</partinfo>
  
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FAST-PETase-t is a dockerin-fused variant of free FAST-PETase (BBa_K4275007). The enzyme carries the same amino acid mutations (N233K, R224Q, S121E, D186H, and R280A) as the former and shares all the common sequences in the CDS, except the C' terminal fusion of a type-I dockerin domain in this enzyme. The catalytic domain of FAST-PETase-t and the dockerin domain are interspaced with a medium-lengthed flexible GS linker (10 aa long) to avoid steric inhibitions. The type-I dockerin domain forms high-affinity non-covalent interaction with type-I cohesins on the cipA scaffoldin.
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FAST-PETase-t is a dockerin-fused variant of free FAST-PETase (BBa_K4275007). The enzyme carries the same amino acid mutations (N233K, R224Q, S121E, D186H, and R280A)[1] as the former and shares all the common sequences in the CDS, except the C' terminal fusion of a type-I dockerin domain in this enzyme. The catalytic domain of FAST-PETase-t and the dockerin domain are interspaced with a medium-lengthed flexible GS linker (10 aa long) to avoid steric inhibitions. The type-I dockerin domain forms high-affinity non-covalent interaction with type-I cohesins on the cipA scaffoldin.
  
 
[[File:GreatBay SCIE--FAST-PETase-t.png|950px]]
 
[[File:GreatBay SCIE--FAST-PETase-t.png|950px]]
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<p align="center"><b>Figure 1</b> The 3D structure of the protein predicted by Alphafold2. </p>
  
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===Usage and Biology===
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==Usage and Biology==
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The improvised FAST-PETase - 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 (FAST-PETase-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 FAST-PETase - 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 (FAST-PETase-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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===Design consideration===
 
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1. The sequence of the first 264 amino acids is identical with the free FAST-PETase (BBa_K4275007).
 
  
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2. A 10 aa long GS linker (GGGGS)2 is appended following the Ser264 residue at the C' terminal of the original sequence.
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==Sequence and Features==
  
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3. 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 8xhis affinity purification tag (HHHHHHHH).
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<partinfo>BBa_K4275008 SequenceAndFeatures</partinfo>
  
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4. DNA sequence is codon-optimized based on the codon-usage table of E.Coli Strain K12.MG1655.
 
  
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==References==
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K4275008 SequenceAndFeatures</partinfo>
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1. Lu, Hongyuan et al. "Machine Learning-Aided Engineering Of Hydrolases For PET Depolymerization". Nature, vol 604, no. 7907, 2022, pp. 662-667. Springer Science And Business Media LLC, https://doi.org/10.1038/s41586-022-04599-z.
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Latest revision as of 00:57, 14 October 2022


FAST-PETase-t

FAST-PETase-t is a dockerin-fused variant of free FAST-PETase (BBa_K4275007). The enzyme carries the same amino acid mutations (N233K, R224Q, S121E, D186H, and R280A)[1] as the former and shares all the common sequences in the CDS, except the C' terminal fusion of a type-I dockerin domain in this enzyme. The catalytic domain of FAST-PETase-t and the dockerin domain are interspaced with a medium-lengthed flexible GS linker (10 aa long) to avoid steric inhibitions. The type-I dockerin domain forms high-affinity non-covalent interaction with type-I cohesins on the cipA scaffoldin.

GreatBay SCIE--FAST-PETase-t.png

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

Usage and Biology

The artificially-designed FAST-PETase - 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 (FAST-PETase-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.


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]


References

1. Lu, Hongyuan et al. "Machine Learning-Aided Engineering Of Hydrolases For PET Depolymerization". Nature, vol 604, no. 7907, 2022, pp. 662-667. Springer Science And Business Media LLC, https://doi.org/10.1038/s41586-022-04599-z.