Difference between revisions of "Part:BBa K4881027:Design"

 
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The construct starts with a T7 promoter, followed by a ribosome binding site and MHETase linked to PETase. PETase encodes for the PETase enzyme, which catalyzes the breakdown of polyethylene terephthalate (PET) to monomeric mono-2-hydroxyethyl terephthalate (MHET). MHETase encodes for the production of the MHETase enzyme, which catalyzes the breakdown of MHET to ethylene glycol and terephthalic acid. These genes are joined together by a 12 amino acid linker containing glycine and serine. This combination seemed to obtain better results in expression according to the paper “Characterization and engineering of a two-enzyme system for plastics depolymerization”, where it was compared with an 8 amino acid linker and a 20 amino acid linker. Finally, before the double terminator, our team decided to add a reporter gene that would express pink chromoprotein as a visual indicator that the bacteria took the plasmid.
  
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References:
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Knott, B. C., Erickson, E., Allen, M. D., Gado, J. E., Graham, R., Kearns, F. L., Pardo, I., Topuzlu, E., Anderson, J. J., Austin, H. P., Dominick, G., Johnson, C. W., Rorrer, N. A., Szostkiewicz, C. J., Copié, V., Payne, C. M., Woodcock, H. L., Donohoe, B. S., Beckham, G. T., & McGeehan, J. (2020). Characterization and engineering of a two-enzyme system for plastics depolymerization. Proceedings of the National Academy of Sciences of the United States of America, 117(41), 25476–25485. https://doi.org/10.1073/pnas.2006753117
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Reference plasmid on Addgene: pCJ190

Revision as of 01:54, 7 October 2023

The construct starts with a T7 promoter, followed by a ribosome binding site and MHETase linked to PETase. PETase encodes for the PETase enzyme, which catalyzes the breakdown of polyethylene terephthalate (PET) to monomeric mono-2-hydroxyethyl terephthalate (MHET). MHETase encodes for the production of the MHETase enzyme, which catalyzes the breakdown of MHET to ethylene glycol and terephthalic acid. These genes are joined together by a 12 amino acid linker containing glycine and serine. This combination seemed to obtain better results in expression according to the paper “Characterization and engineering of a two-enzyme system for plastics depolymerization”, where it was compared with an 8 amino acid linker and a 20 amino acid linker. Finally, before the double terminator, our team decided to add a reporter gene that would express pink chromoprotein as a visual indicator that the bacteria took the plasmid.

References: Knott, B. C., Erickson, E., Allen, M. D., Gado, J. E., Graham, R., Kearns, F. L., Pardo, I., Topuzlu, E., Anderson, J. J., Austin, H. P., Dominick, G., Johnson, C. W., Rorrer, N. A., Szostkiewicz, C. J., Copié, V., Payne, C. M., Woodcock, H. L., Donohoe, B. S., Beckham, G. T., & McGeehan, J. (2020). Characterization and engineering of a two-enzyme system for plastics depolymerization. Proceedings of the National Academy of Sciences of the United States of America, 117(41), 25476–25485. https://doi.org/10.1073/pnas.2006753117

Reference plasmid on Addgene: pCJ190