General Protocols

PCR
For 50 µL reaction:
 Mixed 25 µL of Phusion polymerase master mix + 21.5 µL milli-q water + 1 µL template +
2.5 µL primer solution
o Primer solution was created by mixing 1 µL of each stock primer and 8 µL of milli-q
water

Gel Electrophoresis
 Created 1% gel solution by adding 50 mL 1x TAE buffer to 0.5 g agarose and microwaving
for 90 s
 Added 5 µL SYBR safe dye and poured solution into mold. Allowed to set for about 20
minutes
 Loaded gel with 12 µL of 1 kB ladder
 Mixed 3 µL of 6x loading dye and 15 µL of PCR reaction
o Loaded 15 µL
 Ran gel at 100 V for 30 minutes
 Wearing goggles and lab coat, visualized gel using UV illuminator, covering gel with shield

DPNI Treatment of PCR reaction

 Mixed remainder of PCR reaction and 1 µL of DPN1 enzyme (1 µL per 50 µL PCR reaction)
 Placed in 37°C water bath for 1 h

DNA Purification
 Followed ThermoScientific kit using “PCR cleanup” directions with these modifications:
o Used 20 µL milli-q water instead of elution buffer

E. coli Transformation
 Obtained cells from freezer and thawed on ice
 Added DNA and mixed by flicking. Incubated on ice for 5 minutes
o 1-2 µL for plasmid DNA
o 10 µL for cloning
 Heat shocked cells for 30 s in 42°C water bath
 Incubated on ice for 2 minutes
 Transferred cells to 1 mL of LB media in a 15 mL culture tube. Place in 37°C incubator (with
shaking) for 1 hour
o Plates were removed from fridge and left at RT about 30 minutes before plating
 Plated cells using a spreader (created by holding a Pasteur pipette over the flame and
allowing to bend twice)
o 20 µL for standard transformation
o 100 µL for cloning
 Plates were placed in 37°C incubator (w/o shaking) for 12-14 hours

Inoculating Liquid Culture

 Added 20 mL LB media to 50 mL tube
 20 µL of kanamycin was added
 Used pipette tip to pick colonies. Pipetted up and down
 Taped lid open and placed in incubator for about 6 hours (to about OD 1.0)
 o ODs were measured using spectrophotometer in 1 mL samples, using media as a
blank

Mini-Prep
 Followed instruction from ThermoScientific kit with these notes:
o Cells were pelleted by spinning down culture tubes at 5000 x g for 10 minutes
o 500 µL of resuspension buffer was added to each pellet. 290 µL aliquots were
transferred into two separate tubes per culture
o Collected DNA by adding 35 µL of milli-q water in column

Activity buffer protocol (60°C)

 Set heat bath up to 60°C and allowed to reach temperature
 Moved heat bath to the pH meter station and allowed to re-equilibrate to temperature
 Diluted 10x activity buffer by adding 4 mL into 36 mL of milli-q water
 Once water bath reached temperature, the pH meter temperature probe was placed in the
water bath
 The pH meter was calibrated with a pH 10 standard, followed by a pH 7 standard
 The pH of 1x activity buffer was measured and 5 M NaOH was added dropwise (about 3-4
drops in total) until the meter read pH 7.4
 Sterile filtered using a 0.2-micron filter and a 60 mL syringe
 Tube was wrapped in foil after sterile filtering

General Binding assay protocol

 Weighed out enough solid substrate to create a stock solution that is about 20-40 mM in a
few mLs of activity buffer
 Dilute dthe stock by 10% with milli-Q water
 Created binding buffer by diluting activity buffer by 10% - 36 mL of activity buffer was
added to a 50 mL tube; 4 mL of milli-q water was added to the same tube
 Spectrofluorometer cuvette was washed with milli-q water and dried with acetone and air.
It was then filled with 3 mL of binding buffer. 28 µL of binding buffer was removed from the
cuvette and 28 µL of TN-IYD Tyr was added. The solution was allowed to equilibrate to
60C in the spectrofluorometer for 20 minutes
o To thaw IYD - removed from freezer and allowed to thaw in hand until mostly liquid,
then transferred to ice. Transferred liquid to a 1.7 mL tube and spun down at 14,000
x g for 5 minutes while placing another tube on ice. After spin down, transferred
liquid to pre-cooled tube. Spun down for about 10 seconds to remove any bubbles
 5 Serial dilution tubes were setup, each with 90 µL of binding buffer. Starting from the stock
phenol tube, 10 µL of the previous tube was added to each serial dilution tube and then
pipetted to mix
 Fluorescence measurements were recorded after addition of substrate solution, starting
from solution 1 (least concentrated) and going up to the stock solution. Typically, two
measurements were taken for each solution at 5 µL additions, until the fluorescence began
decreasing significantly
o Max fluorescence value within 522-527 nm range

General Activity Assay Protocol

 Prepared around 30-40 µM substrate solution in activity buffer
 (If extinction coefficient of substrate is known) Performed a 100-fold dilution in the
appropriate solvent and measured UV-Vis
 Set up blank run on HPLC - vial containing 1100 µL of activity buffer in position 2, with a
wash vial containing milli-q water in position 1
 In order to determine retention time of product and substrate prior to reaction, 50 µM
solutions were created of both. 1 mL was loaded into a vial along with 50 µL formic acid.
Vials were placed in the autosampler and HPLC program was ran
 Reactions were set up in 1.7 mL tubes with 900 µL activity buffer in each tube. Volume of
activity buffer corresponding to the appropriate amount of enzyme and substrate was
removed to the reach the following concentrations in 1 mL:
o 250 µM substrate
o 5 µM enzyme
 A blank sample was setup alongside the reaction sample. The blank sample included
substrate, but no enzyme
 The tubes were placed on a 60°C heat block for 5 minutes
 1 mL of 5 % w/v sodium bicarbonate was also placed on the heat block
 Sodium dithionite was weighed out in the meantime
o Each tube requires 5 mg
 Made sure to weigh out more than minimum amount
 Sodium dithionite was dissolved in warm sodium bicarbonate to produce a 5 % w/v
solution
o 100 µL for each 5 mg
 Having a 30-minute timer ready, pipetted 100 µL of dithionite solution. Pipetted up and
down 10 times, while starting timer
o For multiple samples, initiate each reaction 30 seconds apart
 When about 25 minutes passed, mixed internal standard (usually at 0.55 mM) and formic
acid at a 1:1 ratio to form a quench solution
o Each sample will need 100 µL to quench
 55 µL of each was added per sample to the master mix
 As reaction time expired, pipetted 100 µL of quench solution to each sample and pipetted
slowly up and down 10 times
 Vortex samples and spin down at 14,000 x g for 5 minutes
 For samples with enzyme, transferred supernatant into a new tube and spun down again
o After second spin down, transferred supernatant to HPLC vials
 For samples without enzyme, supernatant was directly transferred to HPLC vial
 Run HPLC by placing vials in autosampler
Experimental results

20180702 Binding Assay with 4-bromo-3-hydroxybenzylamine

MW: 202.05
 Added 500 µL binding buffer to ~2 mg 4-Br-3-HBA to create approx. 20 mM solution
 28 µL of TN-IYD Tyr was used

20180705 Binding Assay with 2-bromophenol

MW: 173.009
Density: 1.6235 g/mL

 Made a 30 mM solution of 2BP by adding 32 µL of stock 2BP liquid in 10 mL activity buffer

 1.8 mL were transferred in 0.9 mL aliquots into two new tubes. 100 µL of milli-q water was
added to each tube, to reach a final concentration of 27 mM
 28 µL of TN-IYD Tyr was used

20180706 Binding Assay with 3-bromo-4-hydroxybenzoic acid

MW: 217.02
 Made 27.9 mM solution in activity buffer
 Used 28 µL of TN-IYD Tyr

20180710 Binding Assay with 3-bromo-4-hydroxyphenyl acetic acid

MW: 231.04
 Weighed out 11.2 mg of 3-Br-4-HPA and dissolved in 2 mL of activity buffer to give a
solution that was 23.8 mM
 1.8 mL were transferred in 0.9 mL aliquots into two new tubes. 100 µL of milli-q water was
added to each tube, to reach a final concentration of 21.4 mM
 28 µL of TN-IYD Tyr was added

20180711 Activity Assay with 4-Br-3-HBA

 Prepared about 30 mM solution in 500 µL activity buffer
 Measured UV-vis using extinction coefficient of fluorotyrosine as an estimate
 Tested concentrations: 250 µM and 2.5 mM
o Samples were done in duplicate
 0.55 mM I-Tyr used as internal standard

20180716 Activity Assay with 2BP and 3-Br-4HB

 Weighed 5.8 mg of Br-4-HB for a concentration of 41.99 mM
 Weighed 8.7 mg of
 Made a 30 mM solution of 2BP with 32 µL of stock 2BP in 10 mL activity buffer
 Concentrations: 250 µM and 2.5 mM
o 2BP was not done in duplicate. Br-4HB was done in duplicate
 Internal standard/quench:
o 0.55 mM Cl-Tyr to quench 2BP samples
o 5.5 mM Cl-Tyr to quench 250 µM Br-4HB samples
o 16.84 mM (fridge stock) Cl-Tyr to quench 2.5 mM Br-4HB samples

20180717 Activity Assay with 3-Br-4-HPA

  Weighed 4.6 mg of 4-HPA and dissolved in 1 mL activity buffer to create 30.23 mM solution
 Weighed 8.0 mg of 3-Br-4-HPA and dissolved in 1 mL activity buffer to create 34.63 mM
solution
 Tested concentrations: 250 µM and 2.5 mM
o Samples were done in duplicate
 0.55 mM Cl-Tyr used as internal standard

Binding assay data

Compound Kd (µM)
3-Br-4-HB 170 ± 30
2BP 150 ± 12
4-Br-3-HBA 85 ± 6
4-Br-3-HPA 5.3 ± 0.3

Activity assay data

Compound Rate/[E] (min-1)

2BP 0.029
4-Br-3-HBA 0.190 ± 0.001
4-Br-3-HPA 0.199 ± 0.0008

2BP 0.21
4-Br-3-HBA 0.874 ± 0.027
4-Br-3-HPA 2.23 ± 0.026