Part:BBa_K3130000
SR mutation on PETase
Given its nonpolar nature, PET is practically insoluble in an aqueous solvent, making the kinetic analysis based on concentration difficult. In past studies, researchers often hydrolyze PET’s analogue p‐nitrophenol acetate (pNPA) for collection of kinetic data. The action of the enzyme was carried out under room temperature and measured spectroscopically at 405 nanometers. Under this model, the Vmax and KM values were found to be 3.1±1.1 U and 2.6±0.3 mM respectively under native conditions. To combat this difficulty, a spectrophotometer analysis was carried out to approximate kinetic rate.
Through 3-day cell growth assays in 24-well plates, Team DNHS_SanDiego gathered data to quantitatively measure the effectiveness with which the SR-PETase part (BBa_K3130000) performs its function of breaking Polyethylene terephthalate (PET) into terephthalic acid and ethylene glycol when transformed into K-12 Escherichia coli. As a control group to measure the function of SR-PETase against, the team also created an E. coli strain transformed with an empty vector plasmid, lacking the capability to degrade PET.
The plate assays were incubated and shaken at 35 degrees Celsius and the strains were grown both in the presence and absence of ground PET pellets at a concentration of 8 mg/mL.
Mono-2-hydroxyethyl terephthalate (MHET) is an immediate product of PET degradation and can be detected at an absorbance of 260 nm. So, in order to evaluate cutinase activity, the team measured the absorbance of the cultures’ supernatant at a range of wavelengths from 200 to 300 nm. The following graph depicts this data, with the cultures being diluted 5-fold before relative absorbance was measured:
See graph at: https://2019.igem.org/Team:DNHS_SanDiego/Results
At 260 nm, the supernatant of the E. coli cultures transformed with SR and grown in the presence of PET had a relative absorbance of 3.598. The blank media had an absorbance of 0.201, the empty vector culture grown without PET had an absorbance of 2.245, the empty vector culture grown with PET had an absorbance of 2.214, and the PETase culture grown without PET had an absorbance of 0.233.
This data illustrates that PETase is clearly performing its function of degrading PET into MHET well. The absorbance of the blank media serves as a control value that would indicate the complete lack of molecules detected at 260 nm absorbance, including MHET. The relative absorbance values for the three middle strains (the empty vector strain without PET, the empty vector strain with PET, and the cutinase strain without PET) are all extremely close. These strains are also theoretically lacking in MHET, as they either cannot degrade PET or were cultured without it or both. This means that an average value of approximately 2.250 relative absorbance can be attributed to other molecules released from E. coli that can be detected at 260 nm absorbance. However, the relative absorbance of the SR strain with PET, at 3.634 is greater than 3.598 and the presence of PET was the only difference in the environments of the two groups of SR-PETase bacteria, leading to the conclusion that this difference is a result of MHET produced and SR-PETase is effectively degrading PET.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 513
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 839
Illegal AgeI site found at 269 - 1000COMPATIBLE WITH RFC[1000]
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