Team:UMaryland/Protocols
Protocols
Lysing cells and using PCAU
PcaU fluorescence to measure [PCA]
-Culture BL21 E. coli containing PcaU fluorescent reporter in 50mL flask, 30C, 250rpm
-Prepare multi well plate containing serial dilutions of concentrated PCA solution (in Tris buffer) in wells. Fill gaps between wells with water to prevent drying. Fill wells on edges with water.
-When OD600 = 0.6-0.8, transfer a fixed volume of culture from the flask to each well except for the water wells on edges
-Incubate >6 hours
- Read fluorescence in plate reader at 395 excitation 509 emission
- Optional: spin down plate and resuspend wells in PBS for reduced error
PcaU fluorescence to measure [TPA]
-Prepare multi well plate containing serial dilutions of concentrated TPA solution (in tris buffer) in wells. Fill gaps between wells with water to prevent drying. Fill wells on edges with water.
-Add TPH enzyme mix to each well and incubate at 30C overnight. Recommended 1 volume mix per 10 volumes total
-Proceed to protocol: PcaU fluorescence to measure [PCA]
Harvesting PETase and TPH enzymes
-Incubate 50ml culture of BL21 E. coli containing protein expression plasmid at 37C, 250rpm
-Induce with 1mM IPTG at OD600 = 0.6-0.8
-if TPH enzyme, incubate 5h at 37C at 250rpm. If PETase, incubate 17h at room temp at 250rpm
-Spin down. if PETase, resuspend pellet in 10mM Tris, pH 7.2. Resuspend in TPH Buffer if TPH enzyme.
-Sonicate for 30 min on ice: 5 seconds on, 5 seconds off, amplitude = 20
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umarylandigem@gmail.com
Biology - Psychology Building
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