Difference between revisions of "Team:UMaryland/Protocols"

 
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pNBP Assay
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<br>1. Resuspend the pellet 1:10 (mL resuspension buffer: mL of culture pelleted) with Buffer A (1mL Buffer A for 10 mL cultures)
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<br>2. Sonicate the pellet on ice
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<br>3. Pulse 5 sec on and 5 sec off (20% A) for 3 minutes.
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<br>4. Repeat for 5 cycles. In between cycles, check for foaming.
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<br>5. Centrifuge 10,000 g 20 min to pellet cell debris
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<br>6. Collect supernatant in a clean tube (protein)
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<br>7. Store in freezer until use
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<br>9. pNPB assay, 50 uL sample ADDED FIRST
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<br>10. Pipette out ALL the liquid into a clean 15mL tube
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<br>11. Create a well plate diagram of what is being loaded into which wells (which samples are which, which are replicates etc)
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<br>12. Set up plate reader
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<br>13. Warm up the plate reader, click cancel after it reaches 37 degrees.
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<br>14. Now, add 150 ul pNPB:EtOH:TrisHCl 1:4:95 solution to each wells.
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<br>15. Run the plate
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<br>16. Take data, Get kinetic reads 1,6,11,16,21,26...101. click show, then click the green excel button. Save excel file. Put in Drive
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-Spin down. resuspend pellet in 10mM Tris, pH 7.2 if PETase, TPH Buffer if TPH enzyme.
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-Spin down. if PETase, resuspend pellet in 10mM Tris, pH 7.2. Resuspend in TPH Buffer if TPH enzyme.
 
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Latest revision as of 03:59, 18 October 2018

Template Title Template Title

Protocols
Lysing cells and using PCAU
pNBP Assay

1. Resuspend the pellet 1:10 (mL resuspension buffer: mL of culture pelleted) with Buffer A (1mL Buffer A for 10 mL cultures)
2. Sonicate the pellet on ice
3. Pulse 5 sec on and 5 sec off (20% A) for 3 minutes.
4. Repeat for 5 cycles. In between cycles, check for foaming.
5. Centrifuge 10,000 g 20 min to pellet cell debris
6. Collect supernatant in a clean tube (protein)
7. Store in freezer until use
9. pNPB assay, 50 uL sample ADDED FIRST
10. Pipette out ALL the liquid into a clean 15mL tube
11. Create a well plate diagram of what is being loaded into which wells (which samples are which, which are replicates etc)
12. Set up plate reader
13. Warm up the plate reader, click cancel after it reaches 37 degrees.
14. Now, add 150 ul pNPB:EtOH:TrisHCl 1:4:95 solution to each wells.
15. Run the plate
16. Take data, Get kinetic reads 1,6,11,16,21,26...101. click show, then click the green excel button. Save excel file. Put in Drive
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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