Results

Experimental Summary of Steps Taken in the Cloning of sensor pSB3K3-pmntP-rs-sfGFP:

1. Cut mRFP insert out of pSB3K3-mRFP backbone using EcoRI and SpeI.

pSB3K3-RFP plasmid DNA was purified from an overnight 250ml culture of pSB3K3-RFP in LB media with 50µg/ml Kanamycin using the Omega E.Z.N.A. Plasmid DNA Maxi Kit (catalog #D6922-02) according to manufacturer’s instructions. A final concentration of 87.1ng/µL (1.82 A_260/280, 1.79 A_260/230) was obtained and subsequently used for restriction digest as shown in the following table:

Material	EcoRI	SpeI	Double Digest	Concentration   / Activity
pSB3K3-RFP	11.5 µL	11.5 µL	11.5 µL	41.6ng/µL
10X CutSmart	5 µL	5 µL	5 µL	10x
EcoRI	0.5 µL	0 µL	0.5 µL	20U/µL
SpeI-HF	0 µL	0.5 µL	0.5 µL	20U/µL
Water	33 µL	33 µL	32.5 µL	-
Total	50 µL	50 µL	50 µL	-




The resulting digest was run on a 0.8% gel, and the backbone was gel purified using the Monarch DNA Gel Extraction Kit (#T1020S).

2. HiFi cloning of pmntP-rs-sfGFP geneblock into linearized pSB3K3 backbone

The pSB3K3 backbone and pmntP-rs-sfGFP geneblock were assembled and transformed into NEB5α using the NEB HiFi Assembly Cloning Kit (catalog # E5520S) according to manufacturer’s instructions.

3. Confirmation of target plasmid using EcoRI and SpeI.

Approximately 400ng of plasmid DNA was digested with EcoRI, SpeI-HF or both in order to confirm insertion of the sensor geneblock into pSB3K3. The digest was set up as follows and incubated for 4hr at 37°C in a thermocycler. Subsequently, 25 µL of each reaction was run on a 0.8% agarose gel and imaged on a Fuji LAS 4000.

Material	EcoR1	SpeI-HF	Double   Digest	Concentration / Activity
Plasmid	9.3µL	9.3µL	9.3µL	41.6ng/µL
10x Cutsmart	5µL	5µL	5µL	10x
EcoRI	0.25µL	0µL	0.25µL	20U/µL
SpeI-HF	0µL	0.25µL	0.25µL	20U/µL
Water	35.45µL	35.45µL	35.2µL	-
Total	50µL	50µL	50µL	-




Fig. 2. Restriction digest confirmation of the pSB3K3-pmntP-rs-sfGFP plasmid. 5µL of Purple 1Kb Plus DNA ladder (#N0550S) and 25µL of each sample was run on a 0.8% agarose gel and imaged on a Fuji LAS 4000.




Conclusions:

• The single (EcoRI and SpeI) digests confirm the target plasmid size of 4.5kb, and the double digest showing bands at 2.6kb and 1.8kb serve as confirmation that the plasmid contains both the pmntP-rs-sfGFP insert and pSB3K3 backbone, as desired.
• Additionally, the pSB3K3-pmntP-rs-sfGFP plasmid sequence was confirmed by Sanger Sequencing by GeneWiz.


4. Transformation of pSB3K3-pmntP-rs-sfGFP into MG1655 WT and MG1655 ΔmntR E. coli

Chemically competent MG1655 cells prepared by the 2020 WSU iGEM team and chemically competent MG1655 ΔmntR prepared by the 2022 team were thawed on ice and transformed with pSB3K3-pmntP-rs-sfGFP as follows:

• 100 μL aliquots of cells were thawed from -80°C on ice.
• 2µL of pSB3K3-pmntP-rs-sfGRP plasmid DNA was added to cells and stored on ice for 30 min.
• A pUC19 positive control and a no DNA control were included.
• Tubes were heated at 42 °C for 1 min, and then immediately transferred on ice for 2 min.
• 1 mL of SOC medium was added into the tube and incubated with gentle shaking (250 rpm) at 37 °C for 60min.
• Each was then plated on LB-Kan or LB-AMP (pUC19) plates and cultured overnight at 37 °C.



Result: Transformants of pSB3K3-pmntP-rs-sfGFP were obtained and used to prepare glycerol stocks for future use. The pUC19 plates had lots of colonies, and the no DNA control showed no growth.

Testing of sensor pSB3K3-pmntP-rs-sfGFP

1. Determine if sfGFP is indused in response to MnCl₂

The initial test of the pSB3K3-pmntP-rs-sfGFP sensor was a confirmation that sfGFP protein was produced in response to MnCl₂ treatment. A 1.5M stock solution of Manganese (II) chloride tetrahydrate was diluted roughly into a 10-fold dilution series for use in the assay.




Overnight cultures were set up of:

1. MG1655 E. coli expressing the pmntP-rs-sfGFP sensor
2. A negative control culture of MG1655 ΔmntR (mutant E. coli lacking the MntR transcription factor required for manganese homeostasis) expressing the pmntP-rs-sfGFP sensor]
3. An IPTG-inducible positive control (pET29b-eGFP) known to express eGFP at high levels



Cultures were back-diluted 1:100 in LB+Kan and grown to an OD₆₀₀ of 0.5. The pSB3K3-pmntP-rs-sfGFP sensor cultures were then aliquoted into 1.2 ml cultures for treatment with MnCl₂ as indicated in the table below:

Stock (mM)	Final Concentration (mM)	Culture (mL)	Stock (uL)
1500	136.36	1.2	120
1100	100	1.2	120
110	10	1.2	120
11	1	1.2	120
1.1	0.1	1.2	120
0.11	0.01	1.2	120
0.011	0.001	1.2	120
Water	0	1.2	120




1 mM IPTG was added to the positive control to induce eGFP expression. The cultures were then grown at 37°C, 250rpm. At 3hr, 100µL aliquots were read on a black-shielded 96-well plate (A₆₀₀ and Fluor_485/515). At 3hr, 0.1 OD₆₀₀ equivalent of each sample was collected in 1.5 mL centrifuge tubes and processed for immunoblot to detect GFP levels.

1. Spun 1 OD₆₀₀ equivalent for 20 mins at 13,000 rpm at 4°C.
2. Discarded the supernatant.
3. Resuspended cells in 50 µL loading buffer and boiled for 10 mins at 100°C.
4. Centrifuged at 13,000 rpm for 5 mins. The soluble fraction (supernatant) was collected for immunoblot analysis (Fig.3).


Fig.3: Immunoblot analysis of soluble lysate fraction from MnCl₂-treated MG1655 ΔmntR cells expressing the pSB3K3-pmntP-rs-sfGRP sensor. 5µL of Low MW protein ladder (Thermo Fisher #26616) was run in the first lane followed by 0.01 OD₆₀₀ equivalents from each of the test samples ranging from 0mM (“-C”) to 100 mM MnCl₂-treated. The 10% SDS-PAGE gel was run at 135V and then transferred to a 0.45µm PVDF membrane with 0.35A for 60min and immunoblotted for GFP. Briefly, the blot was blocked 1hr in 5% milk, washed 3 times 15 minutes in TTBS, probed with mouse anti-GFP at 1:1000 in 5% milk overnight at 4°C, washed 3 times 15 minutes in TTBS, probed with goat anti-mouse at 1:5000 in 5% milk for 1 hour at room temperature, and washed 3 times 15 minutes in TTBS. The blot was developed with Western Lightning Plus ECL bioluminescence substrate (2-5min) and imaged on a Fuji LAS 4000.

Conclusions:

• GFP induction by MnCl₂ was observed by western blot of soluble fraction from whole cell lysate.
• There appears to be some carryover from the positive control into the negative lane. Also, it appears that the blocking was insufficient or the GFP antibody dilution used was too low resulting in a lot of non-specific bands on the blot.
• A clear GFP band is present in the 1mM treated sample and a greater level in the 10mM sample indicating the sensor produces sfGFP in response to MnCl₂ treatment.


2. Determine if sfGFP increases in a dose-dependent manner specific to MnCl₂

A₆₀₀ and Fluor_485/515 readings were collected using a BioTek Synergy H1 plate reader for the samples prepared in step 1 above (“Determine if sfGFP is induced in response to MnCl₂”). (Fluorescence was not visible on a standard light box, but was detectable on the plate reader.) Data was analyzed as shown in Fig.4 below, and the fold-change in fluorescence obtained in MG1655 WT and MG1655 ΔmntR E. coli was plotted against the MnCl₂ treatment concentration Fig.5.





Fig. 4: Example calculation of fold-change relative to untreated control. Raw measurements were obtained on the BioTek Synergy H1 plate reader. The average LB media reading was subtracted to obtain blanked A₆₀₀ and Fluor_485/515 values. Next, OD₆₀₀ corrected values were calculated as the ratio of Fluor_485/515 to A₆₀₀. Finally, the fold-change from untreated control was calculated and plotted against the manganese treatment concentration (as shown in Fig.5).



Fig. 5: MnCl₂ dose curve of MG1655 WT and MG1655 ΔmntR cells expressing the pSB3K3-pmntP-rs-mntR sensor. Overnight cultures were diluted 1:100 in LB-Kan and grown at 37oC 250RPM to an OD₆₀₀ of 0.5. Cultures were aliquoted into 1.2ml volumes for treatment with the indicated concentrations of MnCl₂. After 3hr of treatment, A₆₀₀ and Fluor_485/515 readings were collected and the fold-change relative to untreated control were calculated. (No biological replicates used in this initial screen.)

Conclusions:

• When the pSB3K3-pmntP-rs-sfGFP sensor was expressed in WT MG1655 E. coli, an increase in fluorescence was observed above 0.1mM MnCl₂, with a linear increase though 10mM MnCl₂.
• The 0.1mM approximate limit of detection of the current assay is higher than the target 0.009 mM (equivalent to 0.5ppm). Additional assay optimization may help to close this gap.
• No increase in fluorescence was observed when the pSB3K3-pmntP-rs-sfGFP sensor was expressed in MG1655 ΔmntR E. coli lacking the critical manganese responsive mntR transcription factor. This result indicates that the observed fluorescence is being induced in a manner that is dependent on the manganese homeostatic pathway, and suggests that the sensor is performing in a manganese-specific manner.
• Treatments in excess of 10mM inhibited culture growth and were thus omitted from all future experiments.


3. Confirmation that the pSB3K3-pmntP-rs-sfGFP sensor response to MnCl₂ is reproducible.

The manganese treatment assay was repeated with a narrowed range of MnCl₂ based on the results in Fig.5. pSB3K3 vector negative controls were also included as a confirmation of sensor specificity (Fig.6).




Fig. 6: Narrowed range MnCl₂ dose curve of MG1655 WT and MG1655 ΔmntR cells expressing the pSB3K3-pmntP-rs-mntR sensor or control pSB3K3 vector plasmids.Overnight cultures were diluted 1:100 in LB-Kan and grown at 37°C 250RPM to an OD₆₀₀ of 0.5. Cultures were aliquoted into 1.9 mL volumes for treatment with the indicated concentrations of MnCl₂. After 3hr of treatment, A₆₀₀ and Fluor_485/515 readings were collected and the fold-change relative to untreated control were calculated. (No biological replicates used in this initial screen.)

Conclusions:

• The pSB3K3 plasmid controls showed no increase in fluorescence with MnCl₂ treatment.
• MG1655 ΔmntR cultures expressing the pSB3K3-pmntP-rs-mntR sensor also showed no increase in fluorescence with MnCl₂ treatment as observed previously (Fig.6). This data supports our conclusion that the sensor response is specific to the mntR-dependent manganese response.


4. Determination of the time-course of sfGFP production in response to MnCl₂.

To determine the time-course of sfGFP expression in response to MnCl₂, the sensor assay was repeated with biological and technical triplicates with 1mM and 2.5 mM MnCl₂ treatment. A₆₀₀ and Fluor_485/515 readings were taken at 2hr, 4hr, 6hr, 8hr and after overnight incubation (Fig.7).




Fig. 7. Time-course of MnCl₂-induced sfGFP expression in MG1655 WT pBS3K3-pmntP-rs-sfGFP cultures. Overnight cultures were diluted 1:20 in LB-Kan and grown at 37°C 250RPM to an OD₆₀₀ of 0.5. Cultures were aliquoted into 3 mL volumes for treatment with 1 mM or 2.5 mM MnCl₂. At the indicated timepoints, A₆₀₀ and Fluor_485/515 readings were collected and the fold-change relative to untreated (0 mM) control were calculated. 95% confidence intervals are shown.

Conclusions:

• Fold-change in sfGFP increases through 8hr and drops overnight.


5. Determine if sensor performance is improved in M9 minimal medium.

LB has 0.25μM Mn²⁺ that may contribute to observed background fluorescence observed in the no manganese control cultures (Anjem et al 2009). To determine if the use of minimal media could improve sensor performance, MG1655 WT cultures carrying the pSB3K3-pmntP-rs-sfGFP sensor were grown in minimal media lacking manganese and the sensor response to 0.1 mM, 1 mM and 3 mM MnCl₂ was determined (Fig.8).



Fig. 8. Comparison of pBS3K3-pmntP-rs-sfGFP sensor performance in LB and M9 minimal medium. Overnight cultures were grown in LB or M9 medium, diluted 1:100 and grown at 37oC 250RPM to an OD₆₀₀ of 0.5. Cultures were aliquoted into 2 mL volumes for treatment with 0.1 mM, 1 mM or 3 mM MnCl₂. At 2hr of treatment, A₆₀₀ and Fluor_485/515 readings were collected and the fold-change relative to untreated (0mM) control were calculated. 95% confidence intervals are indicated by error bars. All samples run in technical duplicate.

“pTrc-6xHIS-mntR” Sensor Design

Based on the observation that the pSB3K3-pmntP-rs-sfGFP sensor doesn’t work in ΔmntR mutant E. coli and is thus dependent on mntR, it was hypothesized that an inducible mntR plasmid would allow us to optimize sensor performance. A 535 nucleotide geneblock was ordered from Addgene which incorporates 5’ and 3’ homology to the pTrc vector to facilitate HiFi cloning (Fig.9). The geneblock contains the N-terminal 6xHIS tag and the coding sequence for mntR (source). The full promoter has been shown to be more effective than an alternate truncated form which was previously used by the Calgary 2020 iGEM team (BBa_K902073). The manganese-responsive riboswitch (BBa_K902074), sfGFP (BBa_I746916), T1 and T7 terminators (BBa_B0015) were placed downstream of the mntP promoter.

Fig. 9. Description of the pTrc-6xHIS-mntR plasmid.

Experimental Summary of Steps Taken in the Cloning of pTrc-6xHIS-mntR:

The pTrc-VvTs-trGPPS plasmid produced by the 2021 Wright State iGEM team was used as the source of the plasmid backbone used to make the pTrc-6xHIS-mntR plasmid. Plasmid DNA was purified from an overnight 250 mL culture of pTrc-VvTs-trGPPS in LB media with 100µg/ml Ampicillin using the Omega E.Z.N.A. Plasmid DNA Maxi Kit (catalog #D6922-02) according to manufacturer’s instructions. A final concentration of 351ng/µL (1.85 A_260/280, 2.2 A_260/230) was obtained and subsequently used for restriction digest as shown in the following table:

The resulting digest was run on a 0.8% gel and the backbone was gel purified using the Monarch DNA Gel Extraction Kit (#T1020S).

1. HiFi cloning of pmntP-rs-sfGFP geneblock into linearized pSB3K3 backbone

The pTrc backbone prepared above and the 6xHIS-mntR geneblock were assembled and transformed into NEB5α using the NEB HiFi Assembly Cloning Kit (catalog # E5520S) according to manufacturer’s instructions.

2. Confirmation of target plasmid using Nco1 and HindIII.

Approximately 500ng of plasmid DNA was digested with NcoI-HF and HindIII-HF or both in order to confirm insertion of 6xHIS-mntR into the pTrc backbone. The digest was set up as follows and incubated for 4hr at 37°C in a thermocycler. Subsequently, 25 µL of each reaction was run on a 0.8% agarose gel and imaged on a Fuji LAS 4000.

Material	NcoI	HindIII	Double Digest	Concentration / Activity
DNA	6.3 µL	6.3 µL	6.3 µL	79.2/µL
CutSmart	5 µL	5 µL	5 µL	10x
NcoI-HF	0.25 µL	0 µL	0.25 µL	20U/µL
HindIII-HF	0 µL	0.25 µL	0.25 µL	20U/µL
Water	38.5 µL	38.5 µL	38.2 µL	-
Total	50 µL	50 µL	50 µL	-




Fig. 10. Restriction digest confirmation of the pTrc-6xHIS-mntR plasmid. 5 µL of Purple 1Kb Plus DNA ladder (#N0550S) and 25 µL of each sample was run on a 0.8% agarose gel and imaged on a Fuji LAS 4000.




Conclusions:

• The single (NcoI and HindIII) digests confirm the target plasmid size of 4.6kb, and the double digest showing bands at 4.4kb and 230bp serve as confirmation that the plasmid contains both the 6xHIS-mntR insert and pTrc backbone, as desired.
• Additionally, the pTrc-mntR plasmid sequence was confirmed by Sanger Sequencing by GeneWiz.


3. Transformation of pTrc-6xHIS-mntR into MG1655 WT and MG1655 ΔmntR E. coli

Chemically competent MG1655 cells prepared by the 2020 WSU iGEM team and chemically competent MG1655 ΔmntR prepared by the 2022 team were thawed on ice and transformed with pTrc-6xHIS-mntR as follows:

• 100 μL aliquots of cells were thawed from -80 °C on ice.
• 2 µL of pTrc-6xHIS-mntR plasmid DNA was added to cells and stored on ice for 30 min.
• A pUC19 positive control and a no DNA control were included.
• Tubes were heated at 42 °C for 1 min, and then immediately transferred on ice for 2 min.
• 1 mL of SOC medium was added into the tube and incubated with gentle shaking (250 rpm) at 37 °C for 60 min.
• Each was then plated on LB-Kan or LB-AMP (pUC19) plates and cultured overnight at 37 °C.



Result: Transformants of pTrc-6xHIS-mntR were obtained and used to prepare glycerol stocks for future use. The pUC19 plates had lots of colonies, and the no DNA control showed no growth.

Testing of sensor pTRC-6xHIS-mntR induction

1. Determine if mntR is induced in response to IPTG

To confirm that IPTG induces the expression of mntR from the pTrc-6xHIS-mntR plasmid, several different concentrations of IPTG were tested and the induction of mntR was monitored by Coomsasie and immunoblot. Our data showed that 6xHIS mntR protein was expressed in response to 0.1 mM, 1 mM and 10 mM IPTG by 2hr and continuing through an overnight incubation (see Contribution).

2. Determine the effect of mntR expression on the MnCl₂-induced expression of sfGFP from the pSB3K3-pmntP-rs-sfGFP sensor.

To determine the effect of mntR expression on the performance of the pSB3K3-pmntP-rs-sfGFP sensor, two colonies of double transformants (i.e. carrying both pTrc-mntR and pSB3K3-pmntP-rs-sfGFP) were subjected to the MnCl₂ treatment assay (Fig.11). Coomassie and immunoblot of samples from the first colony were performed as confirmation that MntR was induced in these samples (Fig.12).



Fig. 11: Time-course of pSB3K3-pmntP-rs-sfGFP induction in uninduced and IPTG-induced cultures co-expressing the pTrc-6xHIS-mntR plasmid. Overnight cultures of MG1655 WT E. coli expressing the dual plasmid system (i.e. both the pSB3K3-pmntP-rs-sfGFP sensor and pTrc-6xHIS-mntR) were diluted 1:20 in LB-Kan+Amp and grown at 37°C 250RPM to an OD600 of 0.5. Cultures were divided into 25 mL batches and treated with either vehicle or 1mM IPTG for 2hrs at 34°C. Cultures were then aliquoted into 3 mL volumes for treatment with vehicle or 2.5 mM MnCl₂. After 2hr and 4hr of treatment, A₆₀₀ and Fluor_485/515 readings were collected and the fold-changes relative to untreated control were calculated.




Fig. 12. Immunoblot (top) and Coomassie (bottom) of cell lysates from MG1655 ΔmntR E. coli expressing the pTrc-6xHIS-mntR plasmid. Overnight cultures of MG1655 ΔmntR E. coli transformed with the pTrc-6xHIS-mntR plasmid were grown in LB medium with 50 µg/ml Kanamycin, diluted 1:20 and grown to an OD600 of 0.5. The cultures were split and treated with 1mM IPTG or vehicle for 2hrs at 34°C 250RPM followed by 2 additional hours with vehicle or 2.5mM MgCl₂ (as indicated). 5 µL of Low MW protein ladder (Thermo Fisher #26616) was run in the first lane. A positive control of pet29b-6xHIS-GFP was run as a positive control for IPTG induction. 0.1 OD₆₀₀ equivalent of whole cell lysates were run in each lane. Proteins were separated on a 15% SDS-PAGE gel run at 135V and either stained with Coomassie stain or transferred to a 0.45 µm PVDF membrane with 0.35A for 60 min for immunoblot for the 6xHIS tag. Immunoblot was performed using mouse anti-HIS tag (Cell Signaling Technology) at 1:1000 in 5% milk overnight at 4°C and goat anti-mouse IgG at 1:5000 in 5% milk for 1 hour at room temperature. Blots were developed using Lightning Plus ECL bioluminescence substrate and imaged on a Fuji LAS 4000.




Conclusions:

• IPTG induction of pTrc-mntR inhibits the pSB3K3-pmntP-rs-sfGFP sensor. When prior reports that increased mntR levels inhibit the manganese importer (Kauer et al 2014 and Chen et al 2017), it appears likely that the levels of 6xHIS-mntR produced here are sufficient to cause a feedback inhibition of the Mn2+ importer and thus a drop in intracellular Mn²⁺ levels and sfGFP induction.
• As an experimental control, mntR expression was confirmed in IPTG treated samples (Fig.12). Further, addition of 2.5mM MnCl₂ did not interfere with the production of mntR.


3. Determine if the timing of pTRC-6xHIS mntR induction by IPTG affects the performance of the pSB3K3-pmntP-rs-sfGFP sensor.

To determine if the timing of IPTG induction affects sensor performance in MG1655 WT E. coli expressing both the pSB3K3-pmntP-rs-sfGFP and the pTrc-mntR plasmid, the effect of 2hr pre-treatment with 1 mM IPTG and concurrent treatment with 1 mM IPTG was determined (Fig.13).



Fig. 13: 1mM IPTG pre- and concurrent treatment of MG1655 WT E. coli expressing the dual plasmid system (i.e both pSB3K3-pmntP-rs-sfGFP and pTrc-mntR) Overnight cultures of MG1655 WT E. coli expressing both the pSB3K3-pmntP-rs-sfGFP sensor and pTrc-mntR were diluted 1:20 in LB-Kan+Amp and grown at 37°C 250RPM to an OD₆₀₀ of 0.5. Cultures were divided into three batches and treated with vehicle, 1 mM IPTG at 0hr, or 1mM IPTG at 2hr. Cultures were then aliquoted into 3 mL volumes for treatment with vehicle or 2.5 mM MnCl₂. After 4hr of treatment, A₆₀₀ and Fluor_485/515 readings were collected and the fold-changes relative to untreated control were calculated. 95% confidence intervals are indicated by error bars. All samples run in technical and biological triplicate.

Conclusions:

• The uninduced dual plasmid samples expressing both pTrc-6xHIS-mntR and the pSB3K3-pmntP-rs-sfGFP sensor showed a statistically significant fold-change at 4hr of treatment relative to uninduced controls.
• IPTG induction of the pTrc-6xHIS-mntR plasmid blocked pSB3K3-pmntP-rs-sfGFP sensor function when IPTG induction was performed for 2hr prior or done concurrently with MnCl₂ treatment. These data are consistent with prior data (Fig.11).


4. Determine if the co-expression of uninduced pTRC-6xHIS mntR improves the performance of the pSB3K3-pmntP-rs-sfGFP sensor.

Based on our observation that co-expression of the uninduced pTrc-mntR plasmid improved the fold-change in sfGFP produced in response to MnCl₂, we hypothesized that co-expression of the uninduced pTrc-6xHIS-mntR plasmid might improve the sensitivity of the pSB3K3-pmntP-rs-mntR sensor. To test this hypothesis, a 0.01 mM – 2.5 mM dose series of MnCl₂ was used to treat MG1655 WT E. coli expressing the pSB3K3-pmntP-rs-sfGFP sensor alone to uninduced and IPTG induced cultures expression the sensor and the pTrc-mntR plasmid (Fig.14).



Fig. 14: 1 mM IPTG pre- and concurrent treatment of MG1655 WT E. coli expressing the the sensor alone or the dual plasmid system (i.e. both pSB3K3-pmntP-rs-sfGFP and pTrc-mntR). Overnight cultures of MG1655 WT E. coli expressing both the pSB3K3-pmntP-rs-sfGFP sensor and pTrc-mntR were diluted 1:20 in LB-Kan+Amp and grown at 37°C 250RPM to an OD₆₀₀ of 0.5. Cultures were divided into three batches and treated with vehicle, 1 mM IPTG at 0hr, or 1mM IPTG at 2hr. Cultures were then aliquoted into 3 mL volumes for treatment with vehicle, 0.01 mM, 0.1 mM, 1 mM or 2.5 mM MnCl₂. After 4hr of treatment, A₆₀₀ and Fluor_485/515 readings were collected and the fold-changes relative to untreated control were calculated. 95% confidence intervals are indicated by error bars. All samples run in technical duplicate and biological triplicate.

Conclusions:

• As observed previously (Fig.11, Fig.13)As observed previously (Fig.3, Fig.5), IPTG induction of MG1655 WT E. coli cells co-expressing the pSB3K3-pmntP-rs-mntR sensor and the pTrc-6xHIS-mntR plasmid blocked sensor function. None of the measured fold-changes differed significantly from the untreated control (i.e. all 95% CI included 1.0 FC).
• Co-expression of the uninduced pTrc-6xHIS-mntR increased the production of sfGFP in response to MnCl₂ for all MnCl₂ treatment concentrations tested. All measured fold-changes differed significantly from the untreated control (i.e. all 95% CI did not include 1.0 FC).
• The double plasmid system showed a statistically significant induction in response to MnCl₂ treatment down to 0.01 mM MnCl₂, and a dose-dependent induction from 0.01 – 2.5 mM MnCl₂. All measured fold-changes differed significantly from the untreated control (i.e. all 95% CI did not include 1.0 FC)

Overall Findings:

Co-expression of uninduced pTRC-6xHIS mntR with the pSB3K3-pmntP-rs-sfGFP sensor in MG1655 WT E. coli was confirmed as an effective cell-based system for the detection of manganese in water samples down to 0.01 mM (0.5ppm) MnCl₂. This level of detection allows the testing of water samples for manganese contamination near the level at which it is visible (0.05ppm) or deemed unsafe (0.3ppm). Further, the sensor is effective down to the 1mM (5µg/L, 5ppm) practical quantitation limit (PQL) of routine water tests for manganese.