Experiments



Electrophoresis Phenol Red Assay Golden Gate Promoter Assay



Colourimetric Assay E.coli Transformation pH Assay Longer Protocols



Agarose Gel Electrophoresis

agarosegelelectrophoresis

Materials


  • Agarose powder
  • 1X TBE buffer
  • 1% Ethidium bromide
  • NEB 1 kb QuickLoad ladder
  • 4 µl NEB bromophenol loading dye
  • 5-20 µl of your DNA samples
  • Microwave
  • 250 ml conical flask
  • Gel electrophoresis tank
  • Gel tray
  • Well comb and supports
  • Power pack

Protocol


Agarose Gel

  1. Weigh 0.5g of agarose (to make a gel of 1%) and mix with 50 ml 1x TBE buffer in a conical flask
  2. Microwave for 50 seconds or until the agarose is completely dissolved. It is best to heat 30 seconds, stop and swirl towards a boil. Do not over boil and be careful stirring as eruptive boiling can occur. Placing cling film or a lid over the top can help (do not secure the lid, just place it on top).
  3. Allow the agarose solution cool down until you can comfortably hold the flask, if necessary use heat-proof gloves. If it begins to solidify, it can be re-melted. It should take about 5 minutes to cool.
  4. Add 5 μl of Ethidium Bromide. N.B.: Caution the dye is a known mutagen. Wear a lab coat, eye protection and gloves when working with this chemical. Dispose of gloves in appropriate container after.
  5. Pour the agarose into a gel tray with the well comb and supports in place. Pour slowly to avoid bubbles. Bubbles can be popped or pushed to the side using a pipette tip or the comb end.
  6. Let sit at room temperature for 20-30 minutes until completely solidified. Once the gel is set, remove the comb by carefully pulling it up, then remove the supports.

Visualize DNA

  1. Add 1 μl 6x loading dye per 5μl of each of your DNA samples to a PCR tube and mix well by pietting up and down.
  2. Place gel (still on the tray) inside the chamber. Black is negative (cathode) and red is positive (anode). The wells should be facing towards the cathode so DNA will migrate towards the anode. Fill the electrophoresis chamber with 1X TBE buffer so that the buffer covers up the gel and fills the wells.
  3. Gently load 5 μl of 1kb molecular weight ladder (NEB QuickLoad Ladder) into the first lane. N.B.: Maintain positive pressure once sample is pushed out and only release once the pipette tip is out of the buffer to prevent bubbles dislodging the DNA.
  4. Gently load 5-25 μl your samples into the well. It is highly advised to record what you are loading into each well, so that you can label the photo accordingly.
  5. Run the gel at 130V for about 70 minutes or until the dye line is 1-2 cm from the bottom of the gel. N.B: Set yourself a timer and keep on checking on the gel to ensure that the bands will not over run.
  6. Turn OFF the power, disconnect the electrodes and carefully remove the gel and place on a tray.
  7. Using a UV light device (transilluminator) visualise the DNA fragments. Print the photograph and label wells exactly with the samples and the control names, write down the date of the gel. 

Safety considerations


Use safety goggles, face shield and gloves while using Ethidium Bromide and gels/buffers containing it. Dispose of PPE and gels in EtBr-marked bins. Use a lab coat and blue-light glasses or covers to shield your skin and eyes from exposed UV light.



Phenol Red Urease Assay

ph2

Materials


  • PEM buffer(20 mM sodium phosphate buffer pH 7.5, 10 mM β-mercaptoethanol, 20 mM EDTA)
  • UPEM substrate (20 mM sodium phosphate buffer pH 7.5, 10 mM β-mercaptoethanol, 20 mM EDTA, 300 mM urea)
  • Purified jack bean urease (Sigma) for urease activity controls, 1 U/mL in PEM buffer
  • Pooled sterilized human female urine
  • 0.1% (w/v) phenol red in deionized water, sterilized using a 0.22 μm syringe filter
  • 5 M urea in deionized water, filter sterilized
  • 0.9% saline, sterilized by autoclaving
  • 1.5 mL sterile microfuge tubes.
  • 15ml and 50mL sterile conical tubes.
  • 96-well sterile microtiter plate with lid.
  • 25-mL sterile serological pipette.
  • Multichannel pipette (5–50 μL).
  • Water bath + Static and shaking incubators set at 37 °C.
  • Centrifuge and rotor suitable for 50-mL conical tubes.
  • Microcentrifuge
  • Plate reader (visible range, capable of kinetic reads, temperature controlled)

Introduction

Colorimetric urease activity assay utilising the pH altering effect of urease activity to prompt phenol red colour change.

Protocol


Protocol adapted from Richmond's and Yep's paper on the "Quantification of urease activity"

The procedure for this assay involves cell culture growth, microtitre plate preparation, induction of urease and, finally, running the urease assay. To get more details, click on this "Phenol red urease assay link."





Golden Gate Assembly

ph2

Materials


  • T4 DNA ligase
  • 10X T4 DNA ligase buffer
  • Type IIS enzyme
  • Receiver plasmid
  • DNA fragments with Type IIS overhangs
  • MilliQ water

Protocol


  • Set up assembly reactions as follows in a PCR tube:
    • 0.5μL of T4 ligase
    • 2μL of 10X T4 ligase buffer
    • 0.5μL of Type IIS restriction enzyme
    • 100ng of receiver plasmid
    • Equimolar amounts of inserts
    • MilliQ for a total volume of 20μL
  • Mix gently
  • Place the tube on a thermocycler



Pressure promoter Assay

ph2

Materials


  • Overnight cultures of relevant B. subtilis strains
  • Growth medium (M9 used in the relevant paper)
  • Eppendorf tubes (All cultures exposed to non-atmospheric pressure will need a 1mm hole drilled through eppendorf lid)
  • Rack for eppendorf tubes
  • Plate reader
  • Pressurisable vessel
  • 24/96 well plate
  • Ideally, means to control the temperature of the pressurised vessel at physiologically favourable levels

Introduction

Measuring GFP expression when promoted by pressure sensitive promoters. Cultures are grown under pressure before being transferred to a welled plate for absorbance measurements.

Protocol adapted from Guyet's paper on the "Mild hydrostatic pressure triggers oxidative responses in Escherichia coli."

The equations needed to analyse the results obtained from this assay can be accessed through this pressure promoter assay link.


Protocol


  • Dilute overnight cultures to OD600 of 0.04 and grow for 3 hours. Use wild type B. subtilis as a negative control, and B. subtilis engineered expressing GFP via a promoter independant of pressure as a positive control. N.B 3 hours was a time used for E. coli, longer may be required
  • Take 1ml aliquot and place under pressure at 37C for 3 hours. N.B 3 hours was a time used for E. coli, longer may be required
  • After pressure treatment, transfer 3x 200ul to a 24/96 well plate
  • Using a plate reader, monitor the optical cell density at OD600 nm and the GFP signal with excitation 485 nm /emission filter 510 nm. As is typical, use straight media as a blank





CA Colourimetric Assay

ph2

Materials


  • Clarified cell lysate
  • 4-Nitrophenyl acetate (4-NPA)/li>
  • ASSAY SOLUTION (MOPS, Na2SO4, EDTA buffer)
  • DMSO
  • Welled Plate + Plate reader

Protocol


Assay

  • Prepare 182 ul in each well using clarified cell lysate (concentrated to a small volume - this would normally be purified enzyme), 50 mM, pH 7.5 MOPS, 33 mM Na2SO4, 1.0 mM EDTA buffer
  • Add 18 ul of 5 mM 4-NPA solution in 10% DMSO, 90% assay buffer to start the reaction
  • PImmediately start plate reader protocol, measuring absorbance at 348 nm every 12-15s for 5 min

Calculations

  • One unit of activity represents the amount of enzyme catalyzing to produce 1 μmol p-nitrophenol per min under the assay conditions




DH5-alpha Chemically Competent E. coli Cells Transformation

ph2

Materials


  • DH5-alpha Chemically Competent E. coli cells (GoldBio Catalog # CC-101)
  • pUC19 Control DNA, 10 pg/μl
  • Recovery medium (GoldBio Catalog # CC-300)
  • Ampicillin (GoldBio Catalog # A-301ES)
  • LB agar selection plates
  • Microcentrifuge tubes
  • Shaker incubator

Protocol


  1. Remove competent cells from the -80°C freezer and thaw completely on ice (10-15 minutes).
  2. Aliquot 1-5 μl (1 pg-100 ng) of DNA to the chilled microcentrifuge tubes on ice.
  3. When the cells are thawed, add 50 μl of cells to each DNA tube on ice and mix gently by tapping 4-5 times. For the pUC19 control, add 1 μl of (10 pg/μl) DNA to the 50 μl of cells on ice. Mix well by tapping. Do not pipette up and down or vortex to mix, this can harm cells and decrease transformation efficiency. *0.8μl β-Mercaptoethanol (β-ME) can be added to 50 μl competent cells to increase transformation efficiency. Incubate on ice for 10 minutes before adding DNA.
  4. Incubate the cells with DNA on ice for 30 minutes.
  5. After the 30-minute ice incubation, heat shock the cells at 42°C for 45 seconds.
  6. Transfer the tubes to ice for 2 minutes
  7. Add 950 μl of Recovery Medium or any other medium of choice to each tube.
  8. Incubate tubes at 37°C for 1 hour at 210 rpm in a shaker incubator.
  9. Spread 50 μl to 200 μl from each transformation on prewarmed selection plates. We recommend plating two different volumes to ensure that at least one plate will have well-spaced colonies. For the puc19 control, plate 50 μl on an LB plate containing 100 μg/ml ampicillin. Use a sterilized spreader or autoclaved plating beads to spread evenly.
  10. Incubate the plates overnight at 37°C.



CA pH Assay

ph2

Materials


  • Clarified Cell Lysate
  • 0.02 M Tris⋅HCl buffer (pH 8.0. Store in an ice bath at 0-4°C before and during use)
  • Carbon dioxide saturated water (Bubble CO2 gas through 200 ml ice cold water for 30 minutes prior to assay. During saturation process, store water at 0-4°C in an ice bath)

Introduction

The Wilbur-Anderson assay measures the time taken (in seconds) for carbonic anhydrase (CA) activity to cause a pH drop from 8.3 to 6.3 in CO2 saturated water.

Protocol based on this paper on [Carbonic anhydrase assay] and the original 1948 paper on [ELECTROMETRIC AND COLORIMETRIC DETERMINATION OF CARBONIC ANHYDRASE].


Protocol


T0 Determination

  1. Add 6.0 ml of chilled 0.02 M Tris⋅HCl buffer, pH 8.0 to a 15-20 ml beaker. Maintain temperature at 0-4°C and record pH.
  2. Withdraw 4 ml of chilled CO2 saturated water and add to Tris buffer. Immediately start a stop watch and record the time required for the pH to drop from 8.3 to 6.3. Record this time as T0

Enzyme preparation

  1. We wont do this since we're using clarified cell lysate instead of purified enzyme, but we should still keep it cold and work out how it should be diluted. In the original 1948 paper, 'mucosal and brain extracts were made by grinding fresh tissue thoroughly and diluting with about 15 volumes of distilled water'.
  2. If we were going to use purified protein then: Dissolve lyophilized powder at a concentration of 0.1 mg/ml in ice cold water. Store in ice bath prior to use. IMMEDIATELY prior to use dilute suspensions or lyophilized materials to a concentration of approximately 0.01 mg/ml in ice cold water.

Enzyme Activity Assay

  1. Add 6.0 ml of chilled 0.02 M Tris⋅HCl buffer, pH 8.0 to a 20 ml beaker. Maintain temperature at 0-4°C and record pH.
  2. Add 0.1 ml of freshly diluted enzyme. Quickly add 4 ml of CO2 saturated water and record the time required for the pH to drop from 8.3 to 6.3. Record this time as T.

Units Calculation

  1. One unit is defined as a pH drop from 8.3 to 6.3 per minute at 0 °C within a 20 mmol·L−1 Tris-HCl buffer solution saturated with CO2
  2. If we were using purified enzyme, then we could say that units = (2 x (T0 -T1))/(T x mg enzyme in reaction mixture).




Longer protocols


In this section, we have included direct links to the remaining protocols that we have used in our experimentation. These tend to have more details/diagrams or be a combination of other protocols mentioned before.

Type of protocol Purpose
"Berthelot's Urease Activity Assay" Berthelot's reagent is a mixture of phenol and hypochlorite. Ammonia reacts with Berthelot's reagent to form a blue product which then gives samples an OD measurement for colorimetric analysis.
"Christensen's Urease Assay" The urease test is used to determine the ability of an organism to split urea, through the production of the enzyme urease.
"Spizizen medium" Spizizen medium (SM) is a popular minimal medium for the cultivation of B. subtilis.
"Bacillus subtilis Transformation Protocol" Transformation in B. subtilis is achieved by the uptake of donor DNA into recipient cells and the integration of part of this donor DNA into the host chromosome.
"Bacillus subtilis - Clarified cell lysate" Development of a Bacillus subtilis cell-free transcription-translation system for prototyping regulatory elements.
"Q5 Site-Directed Mutagenesis" This kit utilizes the robust Q5 Hot Start High-Fidelity DNA Polymerase along with custom mutagenic primers to create insertions, deletions and substitutions in a wide variety of plasmids.
"Crowley-Gulamani assay" An colorimetric assay to determine urease activity. The hydrolysis of urea to form ammonia causes a pH change which then causes phenol red to change the colour of the solution. Protocol has been designed based on the protocols 'Phenol Red Urease Assay' and 'Urease Assay Protocol (WIP)', both of which are on benchling. The aim of writing this novel protocol is to simplify those which already exist, excluding elements which aren't relevant for our purpose.
"General PCR" Polymerase Chain Reaction (PCR) is a powerful and sensitive technique for DNA amplification.
"DNA ligation" DNA ligation is to join breaks in the phosphodiester backbone of DNA that occur during replication and recombination.
"Miniprep" Miniprep is used to isolate small plasmid DNA from bacteria while limiting contaminating proteins and genomic DNA.
"Gel extraction" Gel extraction is commonly used to isolate DNA from an agarose gel.
"DNA clean up" DNA cleanup is required for efficient removal of primers, nucleotides, enzymes, salts and other impurities from DNA samples prior to use in other purpose.
"Gibson assembly" An efficient and effective method that multiple overlapping DNA fragments can be joined in a single isothermal reaction.
"SDS-PAGE" SDS-PAGE is used to obtain high resolution separation of complex mixtures of proteins.
"Bricks protocol" Bricks protocol.
"Glycerol stock" This protocol is for making cell glycerol stock.
"Improved E. coli ransformation" This protocol is an improved protocol for E. coli transformation.
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