Selective detection of gold using genetically engineered bacterial
reporters
Sebastián Cerminati, Fernando C. Soncini, and Susana K. Checa*
Instituto de Biología Molecular y Celular de Rosario, Departamento de Microbiología,
Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario,
Consejo Nacional de Investigaciones Científicas y Técnicas. Suipacha 531, S2002LRKRosario, Argentina.
Supplementary Material and Methods
Bacterial genetics and molecular biology techniques
The Au-reporter plasmid pPB-GFP (pPB1294, Table I) was constructed as follows: a
285 bp fragment containing the promoter/operator of the golB gene was amplified by
PCR from S. Typhimurim 14028s chromosome using primers PgolB-Fw and PgolB-Rv
(Table SI). The PCR product was digested with XbaI and BamHI and inserted into the
broad-host-range vector pPROBE-NT (Miller et al., 2000), upstream from the gfp gene.
A similar procedure was followed to create plasmid pPA-GFP (pPB1295, Table I). In
this case, the copA promoter region was amplified from Salmonella 14028 genomic
DNA by PCR using the PcopA-Fw/PcopA-Rv pair (Table SI).
Au sensor plasmids pGOLS-GFP (pPB1296) and pGOLS(R)-GFP (pPB1321) that
expresses GolS from its own promoter and GFP under the control of the golB promoter
(Table I) were constructed as follows. A 1800 bp fragment containing the golTS and
golB promoters and the golS gene was PCR amplified from total DNA obtained from
the S. Typhimurium golT strain (PB3298, Checa et al., 2007), using primers PgolTSFw and PgolB-Rv (Table SI). The PCR fragment were treated with XbaI and BamHI
and cloned into the pPROBE-NT plasmid digested with the same enzymes to generate
plasmid pGOLS-GFP. To construct pGOLS(R)-GFP, a 1700 bp fragment including the
golTS promoter and the golS gene were PCR amplified from the Salmonella golT
genomic DNA, as above, using primers PgolTS-Fw and golS-Rv (Table SI). The
fragment and the pPB-GFP plasmid were digested with XbaI and SalI and ligated to
generate the pGOLS(R)-GFP plasmid.
To generate the IPTG controlled golS-expression plasmid, pGOLS-Cm (pPB1322,
Table I), the 490 bp fragment from BamHI-HindIII digested pPB1205 plasmid (Checa
et al., 2007) was cloned into pSU8 treated with the same enzymes. All constructed
plasmids were confirmed by restriction enzyme digestions and sequencing.
The S. Typhimurium PB4836 strain (Table I) was constructed using the Lamda Redmediated recombination (Datsenko and Wanner, 2000; Ellermeier et al., 2002) briefly as
follows. A 1200 bp fragment containing de cat gen was amplified from plasmid pKD3
with primers P1-ges and P2-ges (Table SI) and used to transform a S. Typhimurium
golTS-golB strain (PB3987, Checa et al., 2007) carrying pKD46 plasmid (Datsenko
et al., 2000). Antibiotic resistance from this strain and from the PB5900 strain (Espariz
et al., 2007) were removed using the temperature sensitive plasmid pCP20 carrying the
FLP recombinase (Cherepanov and Wackernagel, 1995) to generate PB4836 and
PB6716 strains, respectively (Table I).
Transgenic E. coli MC1061 golS (PB8820) and MC1061 golTSgolB (PB9453, Table I)
strains were constructed using Lambda Red-mediated recombination (Datsenko et al.,
2000; Ellermeier et al., 2002) briefly as follows. First, fragments of 1700 pb and 3300
pb containing the golS gene with its own promoter or the complete golTS-golB locus
were amplified from the Salmonella golT mutant or the 14028 wild-type strain using
primers pairs PgolTS-Fw/golS-Rv or PgolTS-Fw/golB-Rv (Table SI), respectively, and
separately cloned into the pGEM-T-easy vector (Promega). Then, a 1.1 kb fragment
containing the cat gen was PCR amplified from plasmid pKD3 (Datsenko et al., 2000)
using primers cat-Fw and cat-RV (Table SI), and cloned immediately following either
the golS gene or the golTS-golB cluster in the pGEM-T derivative plasmids. Finally, the
golS-cat or golTS-golB-cat fragments were amplified from the plasmids with primers
P1-gol and P2-cat (Table SI) and used separately to transform E. coli W3110 strain
(Bachamann, 1996) carrying pKD46 plasmid (Datsenko et al., 2000). Chloranphenicol
resistant colonies were selected, checked by direct sequencing and the constructions
were transferred to the E. coli MC1061 strain by P1-mediated transduction (Miller,
1972).
Plasmid and DNA fragments were introduced into bacterial strains by electroporation
using a Bio-Rad apparatus, following the manufacturer’s recommendations.
Metal diffusion assays and Fluorescence microscopy
Metal diffusion assays were performed in SLB (LB without NaCl) agar plates
inoculated with 100 µl of an overnight culture of the Salmonella biosensor. Sterile
celulose filter paper discs (Whatman) containing 10 µl of 60 µM AuHCl4, 1 mM
CuSO4, or 20 µM AgNO3, or sterile water, were placed on the agar surface. After
overnight incubation at 37°C, the fluorescent haloes were observed on a UV
transilluminator.
For fluorescence microscopy, the biosensor strain was grown overnight in the presence
of 40 µM AuHCl4, 1 mM CuSO4 or 10 µM AgNO3, or in the absence of metals. Cells
were harvested by centrifugation, washed and resuspended into an equal volume of the
PBS. For microscopy, agarose pads were prepared on glass slides coated with 1%
agarose dissolved in PBS. Three microlitres of each cell suspensions were placed in the
centre of a coverslip and inverted onto the agarose pad to immobilize bacteria. Bacteria
were viewed at 100X oil immersion in a Nikon Eclipse E800 fluorescence microscope
using the excitation filters FITC-HYQ (excitation 460–500 nm). Images were captured
using SPOT RT 3.0 software, and were further processed for display by using the
Adobe Photoshop software (version 7.0).
References
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sensing of and resistance to gold salts. Mol. Microbiol 63:1307-1318.
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Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. USA 97:6640-6645.
Ellermeier CD, Janakiraman A, Slauch JM. 2002. Construction of targeted single copy
lac fusions using lambda Red and FLP-mediated site-specific recombination in bacteria.
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Legends for supplementary figures
Figure S1. Dose-response curves of the Salmonella biosensor to different metal ions.
Wild-type S. Typhimurium cells carrying the reporter pPB-GFP plasmid were grown for
3 h at 37°C on SM9 medium in the presence of different concentrations of KAu(CN)2
(Au), CuSO4 (Cu), AgNO3 (Ag), ZnCl2 (Zn), CdCl2 (Cd), HgCl2 (Hg), NiSO4 (Ni),
CoCl2 (Co), or FeSO4 (Fe). The increase in fluorescence as induction coefficient (A)
and the final OD600 reached by the cultures (B) were recorded. The data represent the
mean +/- standard deviation of at least eight independent measurements done in
triplicate.
Figure S2. Alternative methods to evidence Au-induced fluorescence. (A) Diffusion
assays on agar plates. Sterile cellulose filter paper discs soaked up with AuHCl4,
CuSO4, AgNO3, or sterile water were placed on the surface of a SLB-agar plate
inoculated with S. Typhimurium 14028s strain carrying the pPB-GFP reporter plasmid.
After incubation, the fluorescent haloes were evidenced in an UV transilluminator. (B)
Fluorescence microscopy. The whole-cell Salmonella biosensor grown in the presence
of 40 µM AuHCl4, 1 mM CuSO4 or 10 µM AgNO3, or in the absence of metals (-), were
observed at the microscope. See the Supplementary Materials and Methods section for
further details.
Figure S3. Dose-response curves of the constructed fluorescent whole-cell biosensors.
E. coli golTSB and golS strains and the wild-type S. Typhimurium strain carrying the
reporter pPB-GFP plasmid were grown until mid exponential phase at 37°C on SM9
medium. Incubation was continued for 3 h in the presence of different concentrations of
KAu(CN)2 (Au) or CuSO4 (Cu). The increase in fluorescence (A) and the final OD600
reached by the cultures (B) were recorded. The data represent the mean +/- standard
deviation of at least eight independent measurements done in triplicate.
Figure S4. Dose-response curves of the transgenic E. coli biosensor to different metals
ions. E. coli golTSB cells carrying the reporter pPB-GFP plasmid were grown for 3 h at
37°C on SM9 medium in the presence of different concentrations of KAu(CN)2 (Au),
AgNO3 (Ag), ZnCl2 (Zn), CdCl2 (Cd), HgCl2 (Hg), NiSO4 (Ni), or CoCl2 (Co). The
increase in fluorescence as induction coefficient (A) and the final OD600 reached by the
cultures (B) were recorded. The data represent the mean +/- standard deviation of at
least eight independent measurements done in triplicate.
Figure S5. Optimization of culture and incubation conditions. (A) Response of
transgenic E. coli golTSB biosensor to Au and Cu in different culture media. Culture
and incubation with metals were done, basically as described in Fig. 1, except for the
carbon source employed for preparation of SM9: 0.2% glucose; 0.2% glycerol; 0.27%
sodium acetate, or 0.32% sodium citrate. KAu(CN)2 (Au) and CuSO4 (Cu) were
employed at a final concentration of 10 µM. (B) Time-dependent induction of GFP
expression by Au ions. Cells cultured in SM9-glucose medium were incubated for 1, 2,
3, 5 and 20 h in the presence of the indicated concentrations of KAu(CN)2. The data
represent the mean +/- standard deviation of at least eight independent measurements
done in triplicate.