Team:ASIJ Tokyo/Parts

Parts

Anderson Promoters

Relative Strength: weak (0.33) BBa_J23110

• Selected as a relatively weak promoter against which to compare the expression of the PETase construct

Relative Strength: moderate (0.58) BBa_J23111

• Selected as a relatively moderately strong promoter against which to compare the expression of the PETase construct

Relative Strength: strong (1) BBa_J23100

• Selected as a relatively strong promoter against which to compare the expression of the PETase construct

Tags

N-OsmY: Fusion proteins with an N-terminal osmY have been shown to successfully secrete proteins of interest out of E. Coli

C-Myc: This C-terminal tag would allow the PETase construct to be more easily detected by Western blot assays for expression

Others

PETase sequence: gene that coded for the PET hydrolase

Plasmid backbone: the pSB1C3 backbone is recommended

High efficiency E. Coli cells: hosted the PETase constructs

Parts Submitted

PETase catalyzes the hydrolysis of PET into terephthalate and ethylene glycol monomers. The N-terminal osmY protein and its signal peptide fused to PETase, with serine-glycine peptide’s repeating intervention, serves to secrete PETase out of the cell (this reacts with PET in the cell's local environment). The Anderson promoters, with relative strengths that precede the fusion protein in the plasmid, serves to constitutively express the fusion PETase, enhancing the degradation of environmental PET. This part offers efficiency in the initial step in the bioremediation of PET: the production of metabolizable monomers (ethylene glycol and terephthalate) through hydrolysis. This part utilizes the pSB1C3 backbone and confers resistance to chloramphenicol.

BBa_K1885001

• Gibson-assembled pSB1C3 with novel fusion osmY-PETase A0.??

BBa_K1885002

• Gibson-assembled pSB1C3 with novel fusion osmY-PETase A0.58

BBa_K1885005

• Gibson-assembled pSB1C3 with novel fusion osmY-PETase A0.33