Part:BBa_K4701303

SpyTag-MHETase

MHETase is one of two enzymes that together form a PET depolymerization pathway [1]. As it has been observed that a fusion between MHETase, and the other enzyme PETase, can improve depolymerization efficiency, we decided to design parts where the two enzymes would be linked by the SpyCatcher/Spytag system [2]. This part contains MHETase coding sequence prefixed with a SpyTag sequence recommended for N-terminus fusions in T7 based expression systems, followed by a linker also recommended by the authors of the SpyCatcher/SpyTag system [3]. The intended use is to form a fusion with part BBa_K4701302 which contains the FAST-PETase sequence domain. The predicted conformation created with ColabFold [4] is shown in Figure 1.

Figure 1: SpyCatcher/SpyTag fusion between this part and BBa_K4701303. (cyan: MHETase, magenta: linkers, yellow: SpyCatcher002, red: SpyTag, orange: FAST-PETase, gray: His-tags)

References

[1] Knott, BC., et al. (2020). Characterization and engineering of a two-enzyme system for plastics depolymerization. Proceedings of the National Academy of Sciences, 117(41), 25476–25485. https://doi.org/10.1073/pnas.2006753117

[2] Zakeri, B., et al. (2012) Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proceedings of the National Academy of Sciencesm, 109(12). https://doi.org/10.1073/pnas.1115485109

[3] Keeble, AH., Howarth, M. (2019) Insider information on successful covalent protein coupling with help from SpyBank. Methods in Enzymology. 617, 443–461, https://doi.org/10.1016/bs.mie.2018.12.010

[4] Mirdita, M., et al. (2022) ColabFold: making protein folding accessible to all. Nature Methods. 19(6), 679–682, https://doi.org/10.1038/s41592-022-01488-1

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]