Part:BBa_K4217005

pTrc-(6xHis)MntR

General description:

This part is an IPTG-inducible version of the BBa_K4217004 mntR metalloregulatory protein. mntR is a regulator of manganese homeostasis in bacteria which was described in 2000 by Que et al. The ¬E.coli mntR was subsequently identified in Patzer et.al. 2001. mntR reacts to intracellular manganese levels to either induce or repress proteins in the manganese homeostatic pathway. MntR induces the expression of the manganese efflux pump mntP, and reduces the expression of the manganese importer mntS. Since this protein is known to respond to the level of intracellular manganese, an IPTG-inducible system that allows titration of mntR levels in E. coli would have potential utility in the experiments requiring the regulation of manganese homeostasis.

“pTrc-6X-HIS-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.1). The geneblock contains the N-terminal 6X-HIS 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. 1. Description of the pTrc-6X-HIS-mntR plasmid.

Experimental Summary of Steps Taken in the Cloning of pTrc-6X-HIS-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-6X-HIS-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:

Material	NcoI	HindIII	Double Digest	Concentration / Activity
DNA	2.8 µL	2.8 µL	2.8 µL	351.1ng/µL
CutSmart	5 µL	5 µL	5 µL	10x
NcoI-HF	0.5 µL	0 µL	0.5 µL	20U/µL
HindIII-HF	0 µL	0.5 µL	0.5 µL	20U/µL
Water	41.7 µL	41.7 µL	41.2 µL	-
Total	50 uL	50 uL	50 uL	-

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 6X-HIS-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 6X-HIS-mntR into the pTrc backbone. The digest was set up as follows and incubated for 4hr at 37°C in a thermocycler. Subsequently, 25 ul 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. 2. Restriction digest confirmation of the pTrc-6X-HIS-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 6X-HIS-mntR insert and pTrc backbone, as desired.
• Additionally, the pTrc-mntR plasmid sequence was confirmed by Sanger Sequencing by GeneWiz.


3. Transformation of pTrc-6X-HIS-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-6X-HIS-mntR as follows:

• 100 μl aliquots of cells were thawed from -80 °C on ice.
• 2 ul 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 60 min.
• Each was then plated on LB-Kan or LB-AMP (pUC19) plates and cultured overnight at 37 °C.



Result: Transformants of pTrc-6X-HIS-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-6X-HIS-mntR induction

1. Determine if mntR is induced in response to IPTG

To confirm that IPTG induces the expression of mntR from the pTrc-6X-mntR plasmid, several different concentrations of IPTG were tested and the induction of mntR was monitored by Coomsasie and immunoblot. Our data showed that 6X-HIS 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. "dual" colonies carrying both pTrc-mntR and pSB3K3-pmntP-rs-sfGFP) were subjected to the MnCl₂ treatment assay (Fig.3). Coomassie and immunoblot of these samples from the first colony were performed as confirmation that MntR was induced in these samples (Fig.4).



Fig. 3: Time-course of pSB3K3-pmntP-rs-sfGFP induction in uninduced and IPTG-induced cultures co-expressing the pTrc-6X-HIS-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-6X-HIS-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 Fuor_485/515 readings were collected and the fold-changes relative to untreated control were calculated.



Fig. 4. Immunoblot (top) and Coomassie (bottom) of cell lysates from MG1655 ΔmntR E.coli expressing the pTrc-6X-HIS-mntR plasmid. Overnight cultures of MG1655 ΔmntR E.coli transformed with the pTrc-6X-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-6X-HIS-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 6X-HIS 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 6X-HIS-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.4). Further, addition of 2.5mM MnCl₂ did not interfere with the production of mntR.


3. Determine if the timing of pTRC-6X-HIS 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.5).



Fig. 5: 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 Fuor_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-6X-HIS-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-6X-HIS-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-6X-HIS 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-6X-HIS-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 dual plasmid system (i.e. the sensor and the pTrc-6X-His mntR plasmids). (Fig.14).



Fig. 6: 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 37oC 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 Fuor_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.3, Fig.5), IPTG induction of MG1655 WT E.coli cells co-expressing the pSB3K3-pmntP-rs-mntR sensor and the pTrc-6X-HIS-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-6X-HIS-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).

Ovearall findings:

Co-expression of uninduced pTRC-6X-HIS 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 well below the level at which it is visible (50ppm) or deemed unsafe (500ppm). Further, the sensor is effective down to the 1mM (5µg/L, 5ppm) practical quantitation limit (PQL) of routine water tests for manganese.

Assembly Compatibility:

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