Measuring arsenite with the help of overnight grown
E. coli DH5(pProbe-arsR-ABS)
(GFP activity)
Principles
Natural resistance to arsenic and antimony
Resistance to arsenite and arsenate is a natural phenomenon, which is present in many
different bacteria. The resistance mechanism functions by pumping any entering arsenite out
from the cell by use of a specific membrane protein complex. Arsenate, which comes into the
cell, is reduced to arsenite by a specific enzyme and then subsequently pumped out again.
The bacteria recognize arsenite molecules within the cell by means of a specific arsenitesensing protein. The protein, called ArsR, not only has a ‘sensing function’, but is also
capable of regulating the synthesis of the biochemical machinerie needed to get rid of
arsenite (i.e., the membrane pump and the arsenate reductase). Hereto, the ArsR protein
binds to the DNA at a specific region. If no arsenite is present, the ArsR protein will inhibit
synthesis of the resistance machinerie. When arsenite enters the cell, it reacts with the ArsR
protein, which ‘falls off’ the DNA and can no longer inhibit formation of the resistance
proteins. The recognition by the ArsR protein is rather specific. Apart from arsenite only
antimonite is known to react with the ArsR protein and to cause release of repression.
The biochemistry of resistance to arsenite and arsenate is very well understood and the
genes necessary for the resistance proteins and the ArsR protein have been isolated. The
DNA sequences are completely known and, therefore, leave room for very detailed and
tailored genetic engineering.
Principle of the test
The principle of the biosensor test is based on the ArsR repressor protein, its interaction with
specific DNA regions and its ability to react with arsenite. By using genetic engineering an
artificial coupling was created between a DNA fragment containing the binding site for ArsR
and a DNA fragment with the gene coding for a green fluorescent protein (stable GFP mutant
(F64L/ S65T)). Both pieces of DNA were then connected to a so-called plasmid DNA, to be
able to introduce and maintain the engineered DNA in a laboratory strain of Escherichia coli.
This E. coli bacterium with the plasmid is the microorganism performing the arsenite test.
Under conditions during which the E. coli cells do not encounter arsenite, the ArsR protein
will inhibit synthesis of GFP (F64L/ S65T). When arsenite enters the cell, the ArsR protein
will release from its binding site, similar as for the original arsenite resistance mechanism.
However, in the arsenite test bacterium release of the ArsR protein from the DNA will result
in synthesis of GFP (F64L/ S65T). How much GFP is being synthesized is dependent on I)
how much arsenite is encountered by the cells, ii) how long the cells are exposed to arsenite,
and iii) the genetic constitution of the E. coli strain used.
GFP fluorescence commonly is quantified and analyzed by fluorimetry. Fluorescence
measured this way is normalized for the number of cells in the sample to obtain an average
fluorescence per cell. Using fluorescence flow cytometry or image cytometry, which are more
sensitive methods, it is possible to measure GFP fluorescence emitted from individual
bacteria.
Construction
The construction of this plasmid was described in Stocker, J., D. Balluch, et al. (2003).
Environ. Sci. Technol. 37: 4743-4750. The strain is kept in our collection under strain number
1598.
pProbe-gfp(tagless)-arsR-ABS (strain 1598)
Figure 3.1: Organization of relevant parts of pProbe-gfp(tagless)-arsR (strain 1594) and pProbegfp(tagless)-arsR-ABS (strain 1598)
Protocol
Material
-
Fluorimeter (Fluostar Galaxy, BMG). Software 4.21-0
-
Greiner 96-well plate
-
Thermomixer
-
centrifuge
Solutions
-
arsenite stock solution (Merck 1.062777 = 50 mM), dilute 100 µl in 5 ml tap water = 1
mM; dilute 100 µl of this in 5 ml tap water = 20 µM.
-
Sterile Luria Broth
-
Minimal medium:
1. Preparations of cell cultures
Escherichi coli DH5 (pProbe-arsR-ABS, strain 1598) is plated on LB agar plus 50 µg/ml
kanamycin sulphate. One colony is inoculated in a 5 ml LB culture with 50 µg/ml kanamicin
sulphate for 14-16 h at 37°C at 200 rpm.
(Optinal: preparation of frozen stocks)
- The next morning 1 ml of this culture is transferred into 50 ml of LB medium. The cells
are incubated at 37°C until an optical density at 600 nm of about 0.6 is reached.
- The culture is put on ice for 15 min, and 10 ml of ice-cold sterile glycerol (87% (vol/vol))
is added and mixed.
-
The mixture is kept on ice while it was divided into 650 µl portions in 1.5 ml Eppendorf
tubes, which are frozen in a dry ice/ethanol bath for 1 min and stored at -80°C.
2. Biosensor incubation
The overnight culture is diluted 0.4 ml to 20 ml in sterile LB/Km, continue incubation
-
for 3 h at 37°C until OD ≈ 0.6.
-
Centrifuge 20 ml at 4'500 rpm for 5 min at room temp. Decant supernatant
-
Resuspend cells in 20 ml minimal medium
(with frozen stocks)
Frozen stocks are thawed in a water bath at 25°C for 2 min right before starting the
-
assay and diluted in LB as follows: 0.65 ml cells plus 5 ml LB. This suspension is
used directly in the assay
Assay mixtures
Assay mixtures contain: 100 µl diluted cell suspension and 100 µl sample (or arsenite
-
stock solution) per well of a 96-well Fluorimeter plate.
-
Standard series can be prepared according to the scheme below.
-
Assays are incubated at 37°C in a rotary shaker (400 rpm).
As concentration
Vol tap water (ml)
(µM)
Volume to add from
Volume to add from
20 µM As stock (µl)
1 mM As stock (µl)
0
0.8
0
0.1
0.8
4
0.2
0.792
8
0.4
0.784
16
1.2
0.752
48
2.0
0.720
80
6.0
0.560
240
20
0.784
16
60
0.752
48
200
0.640
160
Titerplate may look schematically like:
1
2
3
4
5
6
7
8
9
10
11
12
A
M
M
m
m
m
m
m
m
m
m
m
M
B
M
0
0.05 Etc.
M
C
M
0
0.05
M
D
M
0
0.05
M
E
M
0
0.05
M
F
M
0
0.05
M
G
M
0
0.05 0.1
0.2
0.6
1.0
3.0
10
30
100
M
H
M
m
m
m
m
m
m
m
m
M
m
m
M= medium only
Final As concentrations twofold diluted because of mixing with equal volume of cells.
3. GFP (green fluorescent protein) measurement
-
remove lid during measurement
-
setup/path/users/’username’
-
define new folder for current measurements
-
insert in fluorimeter, turn heads to fluorescence
-
change ‚setup/reader configuration’ to ‚fluorescence’
-
select measurement file. Conditions: 1 cycle, 10 flashes, 0.2 delay, cycle time:56,
Exc. Wavelength: 480, emission: 520, Gain: 10
-
activate number of wells to analyse
-
measure
-
open in Excel, ‚update’ and review.
4. Absorbance
-
turn heads to absorbance
-
change ‚setup/reader configuration’ to ‚absorbance’
-
settings: 600 nm, gain: 34
-
(as above) define number of wells to analyse or chose from predefined format
-
measure absorbance (≈ 2 min)
-
open in Excel, ‚update’ and review
5. Calculation
Normalize EGFP fluorescence by the culture absorbance.
Plot normalized EGFP fluorescence as a function of arsenite concentration in µM or µg/L.
This is your standard curve.
Unknown samples can be processed the same way and their fluorescence interpolated in the
calibration curve to derive the "arsenite-equivalent concentration".
Sample pretreatment may be required. See, e.g., the protocols for the luciferase
measurement.