Difference between revisions of "Part:BBa K2835004"
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===Characterization===
 
===Characterization===
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No experimental data was retrieved for this laccase, except for a colony PCR showing the expected insert size. However, theoretical data calculated on the basis of quantum mechanical molecular modelling methods was obtained. For more information on this, please see the iGEM Stockholm 2018 Model wiki page. How the substrate binds to this mutated laccase is shown in Figure 1. For a couple of different conformations, it was concluded that k<sub>cat</sub> and K<sub>m</sub> were approximately constant, and so, they were calculated.
+
No experimental data was retrieved for this laccase, except for a colony PCR showing the expected insert size. However, theoretical data calculated on the basis of quantum mechanical molecular modelling methods was obtained. For more information on this, please see the iGEM Stockholm 2018 Model wiki page. How the substrate binds to this mutated laccase is shown in Figure 1. For a couple of different conformations, it was concluded that k<sub>cat</sub> and K<sub>m</sub> were approximately constant, and so, they were calculated. The linear k<sub>cat</sub> average was computed to 1297,2567 s<sup>-1</sup>.
  
  

Revision as of 12:57, 17 October 2018

His-tagged laccase, mutated to increase the specificity for SMX

This BioBrick codes for a mutated version of a laccase from Trametes versicolor, namely 1GYC (PDB ID). It contains mutated residues at points 162, 332 and 337, from Phenylalanine to Isoleucine. The aim of the mutations was to increase the enzyme's specificity and activity for sulfamethoxazole. It has been codon optimized for Pichia pastoris.

Usage and Biology

This laccase is originally from the fungus Trametes versicolor. Laccases are a class of multi-copper oxidases that fungi use to degrade lignin and be able to grow on wood. Lignin is composed of a cluster of multiple phenolic groups, which are oxidized by laccases. With their natural affinity for phenolic-like structures, laccases have also been shown to degrade a large range of other phenolic compounds. For example, many reports show that laccases can be used to degrade pharmaceuticals - including oestrogens, painkillers and antibiotics. In our project, it has been used to inactivate the antibiotic sulfamethoxazole (SMX), one of the most persistent antibiotics found in the Baltic Sea. This BioBrick is a mutated version of BBa_K2835003 (also created by iGEM Stockholm 2018) with the aim of increasing the enzyme's specificity and activity for SMX. This BioBrick was also codon optimized for Pichia pastoris, the chosen expression host in our project. It was cloned into the Invitrogen pPICZα A vector, which fuses it to the α-factor secretion signal from Saccharomyces cerevisiae, and puts it under control of the methanol-inducible AOX1 promoter.

Characterization

No experimental data was retrieved for this laccase, except for a colony PCR showing the expected insert size. However, theoretical data calculated on the basis of quantum mechanical molecular modelling methods was obtained. For more information on this, please see the iGEM Stockholm 2018 Model wiki page. How the substrate binds to this mutated laccase is shown in Figure 1. For a couple of different conformations, it was concluded that kcat and Km were approximately constant, and so, they were calculated. The linear kcat average was computed to 1297,2567 s-1.


Figure 1: Graphical representation of the enzyme with the substrate, sulfamethoxazole bound (red). The blue residues are the mutated residues mentioned in the text above.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 408
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]