Testing the Bioreporters
Technical material and chemical solutions
- -96-well microtiter plates (PS TC sterile black µclear, Greiner Bio One)
- -Fluorimeter with filter for GFP measurement and for absorbance (FLUOstar Omega (BMG Labtech GmbH, Germany)
- -Field-portable DIY fluorescence detector
- -Incubator for microplates (Thermostar+™ orbital shaker, BMG Labtech GmbH, Germany)
- -Rotary shaker
- -Spectrophotometer (Ultrospec 500 pro, Amersham Biosciences)
- -Cuvets, semi-micro, PS (Ratiolab, Germany)
- -50 ml Falcon tubes
- -4 ml clear glass vials, screw top (Supelco)
- -Solid cap with PTFE/liner 13 mm, for 4 ml vial (Supelco)
- -Tap water
- -Dimethyl sulfoxide (DMSO)
- -Sterile Luria Bertani (LB) broth
- -Antibiotics stock solution: kanamycin (Km) 50 mg/ml
- -MOPS-buffer prepared by mixing:
- 100 ml of 10-fold concentrated buffer stock solution (5 g NaCl, 10 g NH4Cl, 98.36 g MOPS, 0.59 g Na2HPO4*2H2O, 0.45 g KH2PO4 per liter, set pH to 7.0, filter-sterile)
- 2 ml MgCl2 1M
- 1 ml CaCl2 0.1M
- 10 ml Glucose 20%
- to 1 L with milliQ water
- as applicable, the appropriate analyte of interest for setting up the calibration series
- - Mercury
- - Arsenite (Sigma)
- - Octane 10 mM stock solution in DMSO
- Escherichia coli DH5α strain (YOUR TRANSFORMATION for this course) is plated on LB agar plus antibiotics and incubated over night at 37°C to have fresh colonies
- One colony is inoculated into 10 ml LB medium plus 50 µg/ml kanamycin (Km50)
- Grow Escherichia coli DH5α cells over night at 37°C with shaking at 180 rpm
- The next morning, dilute overnight culture (at stationary phase) in fresh medium 50-fold:
200 µl ON culture in 10 ml of LB + 10 µl Km (PREPARE 6 CULTURES)
- Incubate at 37°C and 180 rpm until optical density at 600 nm is 0.4-0.6 (which takes 2-3 hours)
- Measure the culture turbidity (OD600) using a spectrophotometer
- Right before starting the assays, put 3 cultures in 50 ml Falcon tubes (prepare TWO tubes containing 30 ml cell culture)
- Harvest cells by centrifugation at 3’500 rpm for 7 min at room temperature
- Concentrate 2-fold the exponentially growing cells by replacing the LB medium with 15 ml of the MOPS working solution (or tap water)
Calibration Solution Preparation and Biosensor Assays
The specific examples of Mercury and Octane are given below for setting up their calibration curves.
The same principles are used for whichever analyte is of interest in relation to a particular biosensor strain. (The procedure for arsenic has been described previously in this wiki.)
Prepare the calibration series of mercury (Hg) Molar mass of HgCl2 = 271.5 g/mol (⇔ 200.6 g Hg/L ; atomic mass of Hg = 200.59)
HgCl2 25 mM = 6’787.5 mg HgCl2/L (⇔ 5’014.75 mg Hg(II)/L, ≈5 g Hg(II)/L) = 339.4 mg HgCl2/50 ml
Prepare Hg(II) stock solutions in tap water:
- Solution 1 (100 mg Hg/L): 10 µl HgCl2 25 mM (stock solution) + 491.5 µl µl tap water
- Solution 2 (10 mg Hg/L): 100 µl sol. 1 + 900 µl tap water
- Solution 3 (1 mg Hg/L): 100 µl sol. 2 + 900 µl tap water
- Solution 4 (0.1 mg Hg/L): 100 µl sol. 3 + 900 µl tap water
Prepare 10-fold concentrated Hg(II) calibration solutions in Eppendorf tubes according to TABLE 1
Prepare biosensor assays for 4 ml glass vials (pipetting scheme summarized in TABLE 2)
- Put 1.2 ml of biosensor cells suspension in the glass vials according to the layout in TABLE 3
- Put 400 µl of LB rich medium in each glass vial
- Also make one vial that is a 'blank' vial with no biosensor cells, to be used to help correct for density of the biosensor cell suspension, by substituting water for the 1.2ml of cell suspension.
- This is especially necessary for the DIY detector, with its Red LED signal, such that absorbance (OD600) is calculated from -log(Rblank/Rsample)
Making the Hg(II) standard curve
- Add 2 ml tap water in each calibration vial
- Complete to 4 ml with 400 µl of 10x working solutions according to 7 different Hg(II) concentrations, or 400 µl of tap water for the “zero” control
Prepare calibration series of octane
- To note: The AlkST:GFP biosensor should detect many alkanes in general (C6-C12), but the signal in this example will be based upon 'octane equivalents.'
- Also, remember than Octane is very volatile. Do not leave lids off any longer than necessary. Avoiding a large 'head space' is also very useful!
- Prepare 10x dilutions of octane stock solution (final: 1 mM):
by mixing 720 µl DMSO with 80 µl octane 10 mM
- Prepare 100-fold concentrated Octane working solutions in DMSO in 4 ml glass tubes according to the OCTANE CALIBRATION TABLE below.
Preparing the sample assays “1/2”
- Add 2 ml “contaminated” water sample according to the suggestions given in the TABLE 3 (To note, should be at a neutral pH!)
- Complete to 4 ml with 400 µl of tap water
- Prepare a spiked sample assay, which has 2 ml of the sample plus 400 μl of 25 µg/L Hg(II) working solution in sample vials, according to TABLE 3
Preparing the sample assays “1/10”
- Add 400 µl “contaminated” water sample according to the suggestion given in the TABLE 3
- Add 1.6 ml tap water
- Complete to 4 ml with 400 µl of tap water
- Prepare a spiked sample assay, which has 400 µl of the sample, 1.6 ml tap water plus 400 µl of 25 µg/L Hg(II) working solution in sample vials, according to TABLE 3
Alternatively for the Octane Equivalents assays, with the AlkST biosensor
- Complete to 4 ml with 360 µl tap water and 40 µl of DMSO ( also for the 0 of the standard curve )
- for the spiked sample assays, use 40 µl of the 5 µM stock solution, for a 50nM final concentration added to each 'spiked' sample vial.
- Incubate biosensor assay vials at 30°C with shaking at 100 rpm
- After 2h, transfer 200 μl from each glass vial into 3 wells in microtiter plate according to the suggestion given in the TABLE 4
- Measure the fluorescence and absorbance of each well using the fluorimeter
- Record fluorescence of each vial using the Field-portable DIY fluorescence detector
- Plot (in Excel) fluorescence as function of concentration (using the signals obtained from the standard curve vials) and calculate the (-equivalent) concentration in the samples.
Results are presented as the induction coefficients, which are defined as the relative fluorescence units (RFU) of the induced samples divided by that of the untreated sample (background fluorescence)
Preparation of Field Samples
The field sampling protocol is adapted from the manuscript Trang et al. in Environ Sci Tech 2005.
Field samples for arsenic are immediately filtered, acidified and preferably stored at 4C.
To avoid killing the bioreporter cells, the samples must be neutralized.
Any other samples should be checked for pH.
- 200mM Pyrophosphate Buffer (20x concentrated, 10mM final)
- pH meter or paper to confirm the final pH
- Filter the samples (if not filtered already)
- Neutralize the samples using 1:20 dilution (10mM final)
- Check that pH is neutral
Field samples contain more than one metal or compounds that can augment or inhibit the bioreporter molecule production. It is then important to confirm with "spiking" (adding a known amount of the compound to which the bioreporter responds) that there is no such inhibitory or competing compounds.
Pyrophosphate in the Trang paper is used as a chelator to prevent iron from precipitating / interfering with the bioreporter reading.
For some of the water samples brought from India by Shreyasi, more than 10% of the volume of the water sample was needed to bring the pH up to 6.5 or so... Therefore, this volume difference should be taken into account, when the final equivalents (e.g. of arsenic or octane) are calculated based on the observed induction of the biosensor construct!)