• Template DNA
• Forward and Reverse Primers
• Q5 Polymerase
• Deoxynucleotide Triphosphates (dNTPs)
• Nuclease free water
• PCR buffer

1. Create Master Mix using 0.4 μL dNTPs, 10 μL PCR buffer, 33.2 μL nuclease free water, and 0.5 μL polymerase, per sample. Add to each PCR tube.
2. Add 2.5 μL of appropriate forward and reverse primers to each sample.
3. Add appropriate template DNA to each sample.
4. Briefly centrifuge tubes so all liquid collects at the bottom of the tube.
5. Load into thermocycler.
• Annealing temperature is specific to each sample.
• Elongation time is 30 minutes per kbp.

• ZymoPURE P1 (Red)
• ZymoPURE P2 (Green)
• ZymoPURE P3 (Yellow)
• ZymoPURE Binding Buffer
• ZymoPURE Wash 1
• ZymoPURE Wash 2
• ZymoPURE Elution Buffer

1. Centrifuge bacterial sample in a 1.5 mL tube, and discard supernatant.
2. Resuspend cell pellet using 250 μL ZymoPURE P1.
3. Add 250 μL ZymoPURE P2 and immediately mix by inversion. (8-10 times). Incubate at room temperature for 3 minutes.
4. Add 250 μL ZymoPURE P3 (Yellow) and mix by inversion. Invert an additional 5 times after sample turns yellow. Incubate on ice.
5. Centrifuge the lysate for 5 minutes at 16,000 x g.
6. Transfer exactly 600 μL of supernatant into a clean 1.5 mL tube, without disturbing yellow pellet.
7. At 260 μL of ZymoPURE binding buffer to cleared lysate and mix by vortexing.
8. Transfer mixture into a Zymo-Spin II column. Incubate at room temperature for 1 minute and centrifuge at 11,000 x g for 1 minute. Discard flow through.
9. Add 800 μL of ZymoPURE Wash 1 into the column and centrifuge at 11,000 x g for 1 minute. Discard Flow through.
10. Add 800 μL of ZymoPURE Wash 2 to the column and centrifuge at 11,000 x g for 1 minute. Discard flow through. Repeat with 200 μL of Wash 2.
11. Dry spin column at 11,000 x g for 2 minutes.
12. Transfer column to a new 1.5 mL tube. Add 25 μL ZymoPURE Elution Buffer to column matrix, and incubate at room temperature for 2 minutes. Centrifuge at 11,000 x g for 1 minute.
13. Analyze eluted sample using Nanodrop protocol.

1. Create a 100 mL subculture of bacteria of interest.
• Materials: LB, antibiotics
1. Allow to grow to an OD_600nm between 0.6 and 0.8.
2. Incubate on ice to stop growth until next step.
2. Induce subculture using IPTG.
• Materials: 1 M IPTG
1. Add IPTG to culture for a 0.1M concentration.
2. Incubate overnight at 16 °C and 200 RPM.
3. Spin down and add lysis buffer.
• Materials: Lysis buffer (50 mM HEPES, 300 mM NaCl, 10 mM imidazole, pH 8), Ultrapure (Milli-Q Water)
1. Spin at 4000 x g for 3 minutes at 4 °C.
2. Resuspend in 1 mL of ultrapure water (for large cultures).
3. Add 4 mL lysis buffer per 40 mL of culture.
4. Vortex and incubate on ice.
4. Sonicate.
• Place on ice.
• Amplitude: 20%
• Sonicate for 20 seconds, rest for 10 seconds. Repeat 9 times.
5. Centrifuge at 7197 x g for 1 hour at 4 °C.
6. Transfer to cold room.
7. Filter with MCE filter.
8. Add 1 mL of nickel beads solution, and nutate for 1 hr at 4 °C.
9. Wash and elute.
• Materials: DI water, gravity column, wash buffer (50 mM HEPES, 300 mM NaCl, 30 mM Imidazole, pH 8), elution buffer (50 mM HEPES, 300 mM NaCl, 250 mM Imidazole, pH 8), 30% ethanol, collection tubes
1. Load into gravity column. Allow to flow through and sample.
2. Wash using 12 mL of wash buffer. Sample.
3. Wash using 2 mL of wash buffer. Sample.
4. Elute using 12 mL of elution buffer. Sample.
5. Wash gravity column in RT. (Recycle nickel beads by suspending in 30% ethanol. Wash remaining column in DI water and store at 4 °C.)
10. Buffer Exchange.
• Materials: Amicon spin column, exchange buffer, DI water
1. Wash spin column DI water.
2. Transfer elution sample to spin column. (Cutoff depends on size of protein of interest. Save 300 microL for confirmation step.)
3. Spin at 4000 x g for 60 minutes at 4 °C for 0.2 mL
4. Add 12 mL of exchange buffer to column.
5. Repeat 3 and 4.
11. Measure concentration using Bradford protein assay kit (BioRad).
12. Make aliquots of purified protein and store at -80 °C.
13. Confirm protein using SDS-PAGE and, if needed, Western Blot.

• 1x SDS-PAGE Running Buffe
• Laemmli sample buffer
• Beta-mercaptoethanol
• Protein sample

1. Create SDS-PAGE gel (BioRad TGX Stain-Free™ Fast-Cast™ Acrylamide kit)
2. Prepare protein samples:
1. Combine 4.75 microL laemmli sample buffer, 0.25 μL beta-mercaptoethanol, and 5 microL protein sample in a PCR tube.
2. Heat sample at 90-100 °C for 5 minutes.
3. Load gel into electrophoresis cell. Remove comb, and fill chambers with running buffer.
4. Load samples into gel.
5. Run gel electrophoresis at 200V for 39 minutes.
6. Image gel using imager.

1. After SDS-PAGE is completed, electrophoretic transfer of proteins from SDS-PAGE gel to a PVDF membrane was done using BioRad Trans Blot turbo transfer system.
2. After successful transfer of proteins, place the blot in the blocking solution (5% milk in TBS-Tween) overnight at 4 °C.
3. Add 1 μl of Anti-6x His tag antibody (HRP conjugated) to 10 mL of blocking solution and incubate at room temperature for 1 hour.
4. Wash the membrane 3 times with TBS-Tween solution.
5. Add BioRad Clarity western ECL substrate and image using imager under chemiluminiscence and colorimetric modes.

1. Create agarose gel.
• Materials: agarose, 1X TAE Buffer, SYBR Safe Gel Stain
1. Add 0.35 g of agarose and 35 mL of TAE buffer to a flask. Microwave on high for 1 minute.
2. Allow to cool until flask can be comfortably handled, but gel has not begun to solidify. Add 2 μL of SyberSafe dye. Mix.
3. Pour gel into mold. Cover to block out light and allow to solidify.
2. Prepare DNA samples with loading buffer, and add to wells in agarose gel.
3. Run electrophoresis. (Typically run at 120V for 20 minutes.)
4. Image gel using UV lamp or gel imagers.

• Competent cells
• Plasmid sample
• LB
• LB agar plate with ampicillin or carbenicillin

1. Add 1 μL of plasmid sample to a microtube. Gently add 100 μL of competent cells. Also create a sample with no plasmid added as a control.
2. Incubate for 30 minutes on ice. Flick tube every 10 minutes.
3. Heat shock for 1 minute at 42 °C. Place back on ice immediately.
4. Plate sample on LB agar plate with antibiotic added. Allow to grow overnight.
5. Select colonies to culture and test using colony PCR. Confirmed positive colonies may be used for further experiments.

1. Hole-punched amorphous PET films of 6mm diameter are used for PET film-based activity assays. Prior to use, the films are washed with 1% SDS, 100% DMSO, ultrapure water, and 95% ethanol.
2. These washed PET films are subsequently incubated with purified enzymes/E. coli whole cells in 500 μl reaction solution (50 mM Bicine-NaOH, pH 8.5) in a glass test tube at 25 °C accompanied by continuous shaking in an incubator.
3. For analysis, supernatant is withdrawn followed by addition of an equal volume of 100% methanol, heat treatment at 85 °C to quench enzymatic activity and centrifugation to separate E. coli whole cells.
4. The obtained supernatant is filtered using spin filters and used for HPLC analysis.

• 0.01 M FDBz in DMSO
• Enzyme or cell sample (buffer enzyme stored in or wild type of cell sample)
• 10 mM Tris buffer pH 7.4
• Black bottom microplate
• Microplate reader
• Pipette
• 2.5 mL centrifuge tubes

1. Set microplate reader to incubate at 37 °C during preparation. Prepare to take regular fluorescence readings at excitation 488 and emission 523 for 3 hours every 30 minutes.
2. Calculate required volume of enzyme for 0.56 µM final concentration in 100 µL per replicate. Calculate volume of buffer by subtracting result form 100 µL.
3. In a separate tube for each sample, add required volume of buffer followed by enzyme and mix. Repeat for the corresponding negative control of enzyme storage buffer or wild type cell.
4. Transfer 97.5 µL of mixture into each microplate well.
5. Add 2.5 uL of FDBz mixture to each sample.
6. Place microplate into the reader and begin regular measurements.

• Petri dish
• Agar
• LB
• Water
• Antibiotic
• 1 M BHET in DMSO
• Enyme or cell sample
• If cell sample: bunsen burner, L shaped glass spreader, alcohol, PBS buffer

Plate Preparation:

1. Follow normal LB agar media preparation: Mix 2.5 g LB and 1.5 g agar with 200 mL water.
2. Autoclave and place in 55 °C water bath.
3. When cooled, add antibiotics to working concentration and 4 mL BHET solution to final concentration of 20 mM.
4. Pour approximately 10 mL into each petri dish and allow to solidify then store at 4 °C.

Assay:

1. Take plate from storage and allow to warm before making ~1 cm squares on bottom of plate.
2. Add 5 µL of enzyme or cell sample to each square and incubate at 30 °C up to overnight.
3. If cell sample: sterilize spreader by dipping into alcohol and carefully moving over flame of bunsen burner. Allow to cool for ~1 minute before pouring small amount of PBS buffer and spreading to remove top layer of bacteria.
4. Inspect for visual changes in each sample spot.

• Gel from DNA Electrophoresis
• Agarose Dissolving Buffer (ADB)
• ZymoSpin Column and Collection Tube
• DNA Wash Buffer
• DNA Elution Buffer

1. Excise desired DNA fragment from agarose gel used in electrophoresis. Place gel fragment in 1.5 mL centrifuge tube.
2. Add ADB to centrifuge tube. Should be added in a 3:1 volume ADB to volume agarose gel ratio.
3. Incubate at 55 °C for 5-10 minutes, until gel fragment is completely dissolved.
4. Add 200 µL DNA wash buffer to the column. Centrifuge for 30 seconds. Discard flow through and repeat.
5. Dry spin at 12000 x g for 1 minute to remove residual wash buffer.
6. Transfer column to a new centrifuge tube. Add more than 6 microL DNA elution buffer directly to column matrix. Incubate for 5 minutes, then centrifuge for 30-60 seconds.
7. DNA now ready.