Part:BBa_K4217003
mntP-riboswitch-sfGFP-double terminator
General Description:
This composite part is a functional biosensor for manganese in water samples. The part consists of the mntP manganese-responsive promoter, riboswitch and ribosome binding site from E.coli, driving expression of sfGFP. The biosensor was inserted into pSB3K3 by the 2022 Wright State iGEM team and shown to detect manganese chloride down to 0.01mM (500ppm). The sensor performance was enhanced with the co-expression of the pTrc-6X-HIS-mntR manganese regulatory protein part BBa_K4217005.
Experiences/Results
“pSB3K3-pmnpT-rs-sfGFP” Sensor Design
A 1796 nucleotide geneblock was ordered from Addgene which incorporates 5’ and 3’ homology to the pSB3K3 vector to facilitate HiFi cloning (Fig. 1). The geneblock contains the full E.coli mntP promoter (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 terminator (BBa_K902073) were placed downstream of the mntP promoter.
Fig. 1. Description of the pSB3K3-pmntP-rs-sfGFP sensor plasmid.
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 A260/280, 1.79 A260/230) 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).
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.
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 ul of each reaction was run on a 0.8% agarose gel and imaged on a Fuji LAS 4000.
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.
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 follow
Experimental Summary of Steps Taken in the Cloning of sensor pSB3K3-pmntp-rs-sfGFP:</h2>
Conclusions:
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
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:
- MG1655 E.coli expressing the pmntP-rs-sfGFP sensor
- 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]
- A 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:
</style>
1 mM IPTG was added to the positive control to induce eGFP expression. The cultures were then grown at 37°C, 250rpm. At 3hr, 100uL aliquots were read on a black-shielded 96-well plate (A600 and Fuor485/515). 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.
Fig.3: Immunoblot analysis of soluble lysate fraction from MnCl2d 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.
A600 and Fuor485/515 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 Fuor485/515 values. Next, OD600 corrected values were calculated as the ratio of Fuor485/515 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 Fuor485/515 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.
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 Fuor485/515 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.
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 Fuor485/515 readings were taken at 2hr, 4hr, 6hr, 8hr and after overnight incubation (Fig.7).</p>
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 Fuor485/515 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.
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).
| None |