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:
 
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
 
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
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Reference MHETase and PETase plasmid on Addgene: pCJ190

Revision as of 01:56, 7 October 2023

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 MHETase and PETase plasmid on Addgene: pCJ190