Part:BBa_K4681000
pmntP-riboswitch-NanoLuc
The pmntP gene encodes a transcription factor in them manganese homeostatic pathway in E.coli. The ~660bp pmntP promoter displayed a ~5.1-fold induction in response to manganese, in part due to the presence of a conserved riboswitch element (Barrick et. al., 2004). The pmntR promoter-response to manganese was further characterized by Waters et. al. 2011. This composite part incorporates the pmntP promoter and riboswitch placed upstream of a NanoLuciferase reporter.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 255
Illegal XhoI site found at 911 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 768
Overview:
The pmntP-riboswitch-NanoLuc composite part was designed to serve as a biosensor for the detection of manganese contamination in water samples. The part (Figure 1) was synthesized by IDT with EcoRI and SpeI sites flanking the pmntP-riboswitch-NanoLuc coding sequence to enable cloning into a pSB3K3 plasmid backbone for functional testing in both E.coli culture and cell-free assay formats. The sensor produced luminescence in a dose-dependent manner in response to manganese. Luminescence output was sufficient for imaging using a mobile phone. When used in combination with our 3-D printed portable luminescence imaging device (LID) hardware, the sensor provides a fieldable option for users.
Figure 1. Design of the NanoLuc biosensor for the detection of Mn2+ contamination in water. The sensor utilizes the E.coli pmntP promoter including riboswitch and ribosomal binding site (RBS) placed upstream of the NanoLuciferase coding sequence and a double terminator for expression in E.coli.
Functional Testing:
Build: Cloning of pmntP-riboswitch-NanoLuc sensors into pSB3K3 for functional testing: The pSB3K3-pmntP-riboswitch-NanoLuc plasmid was prepared by (1) linearizing the pSB3K3 plasmid backbone by EcoR1/SpeI double digestion, (2) gel purifying the linearized pSB3K3 backbone, (3) HiFi cloning and transformation into NEB5a E.coli. Resulting colonies were screened by restriction digest with EcoR1 and SpeI to confirm presence of the geneblock insert and confirmed by DNA sequencing.
Test 1: Functional testing of the pmntP-riboswitch-NanoLuc sensor in E.coli culture.
To determine if the pSB3K3-pmntP-riboswitch-Nanoluc sensor plasmid responded to MnCl2 in a dose-dependent manner, E.coli cultures carrying the plasmid were grown to an OD600 of 0.5 and treated with 0mM, 0.01mM, 0.1mM, 1mM and 10mM MnCl2 for 6 hours. Samples were diluted with an equal volume of furimazine substrate (NanoGlo Luciferase Assay substrate, Promega N1110). Luminescence was then measured on a BioTek Synergy H1 plate reader. An inducible NanoLuc plasmid (pET-28a(+)::NL) was included to serve as a positive control.
Result: The positive control plasmid yielded strong luminescence (data not shown). The NanoLuc sensor showed a dose-dependent response to MnCl2 (Figure 2). The lowest dose tested, 0.01mM MnCl2, yielded a >2.4 fold increase in luminescence relative to the 0mM MnCl2 control. All test samples yielded measurable luminescence levels within the linear range of the BioTek Synergy H1 plate reader that were stable for at least 15 minutes (data not shown).
Figure 2: Whole-cell testing of the pSB3K3-pmntP-riboswitch-NanoLuc sensor response to MnCl2. Dose-response test NanoLuc sensor to MnCl2. All measurements made without replication (i.e. screening test only).
Test 2: Functional testing of the pmntP-riboswitch-NanoLuc sensor in a cell-free assay format.
Next, we tested the response of the Nanoluc sensor to MnCl2. A no plasmid DNA sample was included as a negative control, and the inducible NanoLuc plasmid (pET-28a(+)::NL) served as a positive control (+C). Luminescence measurements were taken at 2hr, 4hr, 6hr and 24hr to determine the assay time required for sufficient luminescence detection. Reactions were set up in PCR tubes in 12.5µl volumes and incubated at 29C. Luciferase measurements were performed with the NanoGlo Luciferase Assay kit using 1µl sample + 9µl water + 10µl furimazine substrate in a 96-well white plate.
Result: Significant luminescence was observed in the positive control sample, and the Nanoluc sensor samples showed a stable luminescence level 20 minutes beyond substrate addition (data not shown). A consistent fold-change in luminescence from the 0mM MnCl2 control was observed across all timepoints (Figure 3A), indicating a 2-hour assay was feasible. The experiment was repeated 2 additional times, and the combined data showed the NanoLuc sensor responded to MnCl2 with a dose-dependent increase in luminescence from 0.01mM - 1mM MnCl2. (Figure 3B).
Figure 3: Cell-free testing of the pSB3K3-pmntP-riboswitch-NanoLuc sensor response to MnCl2. (A) NanoLuc sensor dose response to MnCl2 with fold-change values calculated as the change in luminescence relative to the untreated (0mM MnCl2) control measured at 2, 4, 6 and 24hr. (B) Dose-response test NanoLuc sensor to MnCl2 at the 2-hour time point. Error bars in panel C indicate the mean ± 1 standard error.
Test 3: Estimation of the limit of detection for the pSB3K3-pmntP-riboswitch-Nanoluc sensor response to MnCl2 in cell-free assay.
The response of the Nanoluc sensor to an extended range of MnCl2 doses was measured to estimate the limit of detection for the assay (Table 8). A no DNA sample was included as a no plasmid DNA negative control (-C), and the inducible NanoLuc plasmid (pET-28a(+)::NL) served as a positive control (+C). Cell-free reactions were set up and measured as in Test 2 above.
Results: The NanoLuc sensor generated luminescence in a dose-dependent manner in response to 0.0025 - 0.5mM MnCl2 (Figure 4A, adjusted R2 = 0.9949). The limit of detection (LOD) of the NanoLuc assay was calculated using the formula LOD = BLSD + 3SD control sample + 3 standard deviations (cite). We converted the LOD luminescence limit (677,380) to mM MnCl2 using the formula LOD(mM) = mx+ b = 1,135,245.46 * 677,380.25 + 595,434.71 = 0.007mM MnCl2. The LOD of 0.007mM MnCl2 corresponds to 0.4ppm. This LOD is below the 0.5ppm exposure limit suggested by O’Neal et. al. 2015 [1] and equal to the World Health Organization limit of 0.4ppm established in 2004 [2].
Luminescence of these samples was also measured in a 96-well white plate using an iPhone 11 (30 second exposure, ISO 8000, 26mm f1.8, 12MP) (Figure 4B), and the luminescence emitted was sufficient for imaging.
Figure 4: Dose response of the pSB3K3-pmntP-riboswitch-NanoLuc sensor to 0.0025 - 0.25mM MnCl2 by cell-free assay. (A) Dose-response was measured 2 hours after assay start and imaged on a BioTek Synergy H1 plate reader. Error bars indicate +/- 1 standard deviation. (B) Luminescence measured by iPhone imaging.
Test 4: Demonstrate the capability of the NanoLuc sensor to detect manganese in water samples.
Several raw and filtered water samples were obtained from grab samples taken as part of Dr. Stephen Jacquemin’s (WSU Lake Campus) routine Grand Lake St. Marys watershed monitoring work. These samples were frozen prior to receipt for testing in our assay. All samples were imaged using the Biotek Synergy H1 plate reader and the Luminescence Imaging Device (LID) hardware described in the Hardware Wiki page. The LID hardware is a 3-D printed device that serves as a portable darkroom. It has a holder for a mobile phone and test samples to ensure consistent imaging from run to run. Cell-free reactions were set up and measured as in Test 2 above.
Results: Measurements made using the BioTek Synergy H1 plate reader yielded a linear dose-dependent increase in luminescence in response to manganese from 0.01mM - 0.5mM (r2 = 0.99, Figure 5A). The linear regression of the standard curve was performed to calculate the manganese levels for the water samples (Table 1, “Plate Reader” data).
In parallel, manganese levels from the same reactions were measured using the Luminescence Imaging Device (LID) hardware and an iPhone 11 (described in the Hardware Wiki, Figure 5B). The resulting light image (bottom) shows all 8 sample tubes were clearly visible. The MnCl2 samples showed the expected dose dependent increase in luminescence (middle), clearly evident in the line profile trace (top) and linear regression (Figure 5C, r2 = 0.987).
Figure 5: Comparison of manganese measurements made on a plate reader and the LID device. (A) MnCl2 dose curve measured on a BioTek Synergy H1 plate reader. (B) Images collected using the Luminescence Imaging Device (LID) hardware including a light image of the tubes in place, the luminescence image, and a pixel intensity trace of the luminescent image. (C) Standard curve determined from iPhone imaging measurement in panel B quantitated using ImageJ.
Manganese levels in the water samples #1003, #1004 from Mike Ekberg, Miami Conservancy District, and the cold water and wetland samples from Stephen Jacquemin, Wright State University Lake Campus were calculated using the standard curve in Figure 5C. The calculated mM levels were converted to ppm and are listed in (Table 1, LID). All water samples tested exhibited manganese levels were in the measurable range for our sensor (i.e. higher than the limit of detection for the NanoLuc assay).
The ppm values measured using the plate reader and the LID varied were not exactly similar, but were above the minimum ppm limit of 0.4ppm [2].
Table 1: MnCl2 measurements from water samples made using a plate reader and the LID hardware. Plate reader: BioTek Synergy H1 plate reader; and LID: the luminescence imaging device and iPhone 11.
Conclusion:
The pmntP-riboswitch-NanoLuc composite part responds to manganese in a dose-dependent manner with a limit of detection of 0.4ppm, a level below the 0.5ppm exposure limit suggested by O’Neal et. al. 2015 [1] and equal to the World Health Organization limit of 0.4ppm established in 2004 [2]. When used in combination with the 3-D printed portable luminescence imaging device (LID) hardware and imaged using a mobile phone, the sensor provides a fieldable option for users.
References:
[1] O'Neal SL, Zheng W. Manganese Toxicity Upon Overexposure: a Decade in Review. Curr Environ Health Rep. 2015 Sep;2(3):315-28. doi: 10.1007/s40572-015-0056-x. PMID: 26231508; PMCID: PMC4545267. [2] Guidelines for drinking-water quality: Fourth edition incorporating the first and second addenda. (2022). World Health Organization.
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