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Characterization and engineering of a two-enzyme system for plastics depolymerization.
Knott BC, Erickson E, Allen MD, Gado JE, Graham R, Kearns FL, Pardo I, Topuzlu E, Anderson JJ, Austin HP, Dominick G, Johnson CW, Rorrer NA, Szostkiewicz CJ, Copie V, Payne CM, Woodcock HL, Donohoe BS, Beckham GT, McGeehan JE
Proc Natl Acad Sci U S A. 2020 Oct 13;117(41):25476-25485. doi: 10.1073/pnas.2006753117. Epub 2020 Sep 28. PubMed Article

Plasmids from Article

ID Plasmid Purpose
162665pCJ136pET-21b(+) based plasmid for expression of MHETase from Ideonella sakaiensis 201-F6 (Genbank GAP38911.1), codon optimized for expression in E. coli K12, with C-terminal His tag.
162666pCJ189pET-21b(+) based plasmid for expresion of MHETase (Genbank GAP38911.1) linked to PETase (Genbank GAP38373.1) from Ideonella sakaiensis by an 8 residue Gly/Ser linker; codon optimized for expression in E. coli K12, with C-terminal His tag.
162667pCJ190pET-21b(+) based plasmid for expresion of MHETase (Genbank GAP38911.1) linked to PETase (Genbank GAP38373.1) from Ideonella sakaiensis by a 12 residue Gly/Ser linker; codon optimized for expression in E. coli K12, with C-terminal His tag.
162668pCJ191pET-21b(+) based plasmid for expresion of MHETase (Genbank GAP38911.1) linked to PETase (Genbank GAP38373.1) from Ideonella sakaiensis by a 20 residue Gly/Ser linker; codon optimized for expression in E. coli K12, with C-terminal His tag.
162669pCJ196pET-21b(+) based plasmid for expression of MHETase from Ideonella sakaiensis 201-F6 (Genbank GAP38911.1), codon optimized for expression in E. coli K12, with C-terminal His tag, incorporating catalytic mutation, S225A
162670pCJ197pET-21b(+) based plasmid for expression of MHETase from Ideonella sakaiensis 201-F6 (Genbank GAP38911.1), codon optimized for expression in E. coli K12, with C-terminal His tag, incorporating S131G mutation.
162671pCJ198pET-21b(+) based plasmid for expression of MHETase from Ideonella sakaiensis 201-F6 (Genbank GAP38911.1), codon optimized for expression in E. coli K12, with C-terminal His tag, incorporating F495I mutation.
162672pCJ199pET-21b(+) based plasmid for expression of the putative MHET hydrolase from Comamonas thiooxydans (Genbank WP_080747404.1) with C-terminal His tag, codon optimized for expression in E. coli K12.
162673pCJ200pET-21b(+) based plasmid for expression of MHETase from Ideonella sakaiensis 201-F6 (Genbank GAP38911.1), codon optimized for expression in E. coli K12, with C-terminal His tag, incorporating S136C and a sequence Tyr75:Phe104 to introduce a 6th disulfide bond as in AoFaeB-2.
162674pCJ201pET-21b(+) based plasmid for expression of MHETase from Ideonella sakaiensis 201-F6 (Genbank GAP38911.1), codon optimized for expression in E. coli K12, with C-terminal His tag, incorporating C224W and C529SS mutations.
162675pCJ202pET-21b(+) based plasmid for expression of PETase from Ideonella sakaiensis 201-F6 (Genbank GAP38373.1), codon optimized for expression in E. coli K12, with C-terminal His tag, incorporating W159C and S238C mutations.
162676pCJ205pET-21b(+) based plasmid for expression of MHETase from Ideonella sakaiensis 201-F6 (Genbank GAP38911.1), codon optimized for expression in E. coli K12, with C-terminal His tag, incorporating C224A an
162677pCJ204pET-21b(+) based plasmid for expression of MHETase from Ideonella sakaiensis 201-F6 (Genbank GAP38911.1), codon optimized for expression in E. coli K12, with C-terminal His tag, incorporating C224H and C529F mutations.
162678pCJ203pET-21b(+) based plasmid for expression of the putative MHET hydrolase from Comomonas thiooxydans (Genbank WP_080747404.1) with deletion of the predicted signal peptide (Phe2:Ala75)
162679pCJ206pET-21b(+) based plasmid for expression of MHETase from Ideonella sakaiensis 201-F6 (Genbank GAP38911.1), codon optimized for expression in E. coli K12, with C-terminal His tag, incorporating the E226T mutation to the putative lipase box.
162680pCJ207pET-21b(+) based plasmid for expression of the putative MHETase from Hydrogenophaga sp. PML113 (Genbank WP_083293388.1) with C-terminal His tag, codon optimized for expression in E. coli K12.
162681pCJ208Lidded variant of PETase from Ideonella sakaiensis 201-F6 (Genbank GAP38373.1) removing a seven-residue loop of PETase (Trp185:Phe191, PETase numbering) and replacing it with Gly251:Thr472 from MHETase; codon optimized for expression in E. coli K12, with C-terminal His tag.
162682pCJ209Lidless variant of MHETase from Ideonella sakaiensis 201-F6 (Genbank GAP38911.1) replacing Gly251:Thr472 of MHETase with the 7-residue loop (Trp185:Phe191, PETase numbering) of PETase , codon optimized for expression in E. coli K12, with C-terminal His tag.
162683pCJ210pET-21b(+) based plasmid for expression of MHETase from Ideonella sakaiensis 201-F6 (Genbank GAP38911.1), codon optimized for expression in E. coli K12, with C-terminal His tag, incorporating S136C, G489C, S530C, and a sequence Tyr75:Phe104 to introduce a 6th and 7th disulfide bond as in AoFaeB-2.
162684pCJ211pET-21b(+) based plasmid for expression of the putative MHETase from Hydrogenophaga sp. PML113 (Genbank WP_083293388.1) with deletion of the predicted signal peptide (Lys2:Gly19), with C-terminal His tag, codon optimized for expression in E. coli K12.
162685pCJ217pET-21b(+) based plasmid for expression of MHETase from Ideonella sakaiensis 201-F6 (Genbank GAP38911.1), codon optimized for expression in E. coli K12, with C-terminal His tag, incorporating two point mutations, G489C and S530C, to introduce a 6th disulfide bond (from PETase).
162686pCJ220pET-21b(+) based plasmid for expression of MHETase from Ideonella sakaiensis 201-F6 (Genbank GAP38911.1), codon optimized for expression in E. coli K12, with C-terminal His tag, incorporating lid deletion (Gly251:Thr472) and C224W and C529S mutations from PETase active site.
162687pCJ221pET-21b(+) based plasmid for expression of MHETase from Ideonella sakaiensis 201-F6 (Genbank GAP38911.1), codon optimized for expression in E. coli K12, with C-terminal His tag, incorporating lid deletion (Gly251:Thr472) and C224H and C529F mutations from PETase active site.

Antibodies from Article