Insecticides are one of the most significant sources of organic phosphate pollution in water bodies. As the active metabolite of organophosphate insecticide, paraoxon could result in neurotoxic poisoning in animals. Our implementation hardware included a filtering device that contains bacteria engineered to overexpress the oph gene, which encodes the enzyme organophosphate hydrolase (OPH).

In order to prove the ability of OPH to hydrolyze and, thus, detoxify paraoxon, we utilize the pNP sensor as the specified switch.

To determine the effect of the paraoxon hydrolysis reaction, we detected the amount of the reaction’s product, p-nitrophenol (pNP) with the pNP sensor. We designed the biosensor by transforming the pNP sensor plasmid and enzyme plasmid into the biological system E. coli BL21(DE3). The enzyme plasmid contains the original lac operon and the subcloned oph gene. The sensor plasmid contains the subcloned GFP (green fluorescent protein) gene and pNP mut1-1 gene, which encodes for the transcription factor protein to enhance downstream GFP expression when induced by pNP (Jha et al. 8495).

The biosensor functions fully under exposure to both IPTG and paraoxon in the environment. With the induction of IPTG, the repressor protein is prevented from binding to the gene, allowing the transcription of the OPH protein. OPH then hydrolyses paraoxon, producing p-nitrophenol (pNP), which would bind to the activator protein pNP mut1-1, leading to the increased transcription of the GFP protein and, thus, causing the emission of green fluorescence (Jha et al. 8495). By measuring the intensity of GFP fluorescence, we can detect the detoxification of paraoxon and the level of inorganic phosphate production.

To determine the standard curves for the positive correlation between pNP and GFP fluorescence intensity for further experiments, we titrated E. coli BL21 (DE3) carrying pNP sensor with varying concentrations of pNP. The concentrations and the resulting fluorescence intensity are mapped out as the standard curve for experimental references.

Experimental work 2: Paraoxon Hydrolysis E. coli BL21 (DE3) carrying pET22b::OPH in presence of IPTG is treated with paraoxon. The rate of paraoxon hydrolysis by OPH can be calculated from the pNP production to prove the target protein's effectiveness.

The data of the preliminary experiment, which aimed to test the transcriptional factor induced by pNP, showed no significance. The results were inconsistent with the research paper on which the experimental design was based. For more information on the engineering of the pNP sensor gene, please visit the Engineering Success page .

Therefore, we redesigned a more direct method to test the degradation of paraoxon by OPH. The experimental design was based on the hypothesis that paraoxon (transparent) would be hydrolyzed to produce diethyl phosphate (transparent) and pNP (yellow, absorbance peak at 410 nm by spectrophotometry). We detected the production level of pNP with spectrophotometry to test the percentage of paraoxon degradation by OPH that could hydrolyze in a certain period of time. We performed two experimental works: the amount of pNP detection at the various time points (pNP conc. v.s. Time) and in the presence of different IPTG concentrations at a fixed time (pNP conc. v.s. IPTG conc.), respectively, to determine the optimal factors of OPH degradation activity.

To ensure the function of OPH before introducing other variables and performing complete experiments with all control groups, we designed and conducted a preliminary experiment. 3 groups of BL21 (DE3) bacteria and 3 groups of BL21 (DE3) bacteria engineered with OPH gene are cultured with medium only as the negative control, paraoxon, and pNP, respectively. The engineered bacteria are then induced by IPTG for protein expression. To test if the products of the hydrolysis reaction would be released into the environment, we measured the samples from the bacterial suspension and supernatant after centrifugation. We then deducted the background data (negative control) from the absorbance and divided the result by the optical density of the bacteria to test the level of pNP produced (and, therefore, the paraoxon degraded) per 10^7 CFU of bacteria added, indicating the ability of paraoxon degradation by each bacteria.

The absorbance of the paraoxon solution increased 6.720481928 A.U. (highlighted in green) after the reaction with BL21 (DE3) engineered with the oph gene, whereas that of the paraoxon solution reacted with BL21(DE3) only increased 0.2323266987 A.U. (highlighted in green). The significant increase in the production of pNP proves the ability of our target protein OPH to hydrolyze paraoxon. Based on the successful results of our preliminary experiment, we further designed experimental works 1 and 2 to investigate the optimal reaction time and IPTG induction level, respectively, that would allow OPH to reach its highest enzyme expression and activity.

After observing that reaction with bacteria alters the absorbance of pNP solution significantly, we constructed a standard curve that measures the absorbance at 410 nm versus the concentration of pNP solution cultured with 0.6 O.D. bacteria for 6 hours.

We aimed to test the optimal reaction time for OPH enzyme activity with experimental work 1. All solutions are prepared with 500 μM PXN/pNP chemical, 100 μM IPTG, and 0.6 O.D . bacteria. The experimental groups are designed as below:

The absorbance at the wavelength of 410 nm and 600 nm in each group was measured after 1 hr, 2 hrs, 3 hrs, 4 hrs, 5 hrs, 6 hrs, and 24 hrs of reaction to test the pNP production level and the concentration of bacteria over time.

The result of experimental work 1 shows that pNP concentration increases rapidly in the first 5 hours and grows steadily afterward.

We aimed to determine the optimal concentration of IPTG induction for OPH enzyme expression with experimental work 2. Solutions are prepared with 500 μM PXN/pNP chemical, 0.6 O.D. bacteria, and various concentrations of IPTG. The experimental groups are designed as below:

The absorbance values at the wavelength of 410 nm and 600 nm were measured after 6 hrs and 23 hrs of reaction to test the pNP production level and the concentration of bacteria with increasing concentration of IPTG.

The results of experimental work 2 demonstrated that after 23 hours, the experimental group induced by 250 μM IPTG reached the highest level of pNP concentration, thus proving that 250 μM IPTG is the optimal induction for the highest OPH protein expression.

Inorganic phosphate (Pi) accumulated in the sediments of aquatic environments is the primary reason for eutrophication. In order to decrease the amount of inorganic phosphate in water bodies, our team implemented a device that contains specially engineered E. coli that expresses the RNA of antisense PhoU (AsPhoU) thus hindering the expression of PhoU and enhancing phosphate transportation. Through our engineering process, these bacterial bodies can absorb more Pi from the environment.

PstSCAB is a high-affinity phosphate transporter protein in E. coli . Pho regulon is a common bacterial regulatory system determining and managing intracellular Pi concentration through the regulation of the PstSCAB transporter protein. The regulon consists of a histidine kinase sensor protein – PhoR – on the inner membrane and a response regulator – PhoB – on the cytoplasmic side of the membrane of a prokaryotic cell. The way PhoB regulates the simultaneous expression of PstSCAB and PhoA depends on the process by which PhoU binds onto PhoR. PhoU, a metal-binding protein, detects the intracellular concentration levels of Pi. When the environment undergoes a decrease in Pi concentration, PhoU dissociates from PhoR and the PstSCAB transporter. This, in turn, promotes the transportation of Pi and increases PhoA expression, which absorbs more Pi into the cell and enhances the phosphorylation of PhoB. On the other hand, when the phosphate level is high, PhoU would be activated to block its absorption, meaning that the transportation by the PstSCAB protein and the expression of PhoA would be inhibited.

A previously published paper demonstrates that PhoU knockout leads to a halt E. coli growth (Haldimann). Therefore, we designed the mRNA for AsPhoU hindering phoU expression to increase the amount of Pi that bacteria could transport, thus lowering the concentration of Pi in aquatic environments. AsPhoU cell expresses antisense PhoU RNA under arabinose-control promoter (pBAD) and thus binds to the mRNA of PhoU, hindering ribosome binding to decrease phoU translation. The inhibition of PhoU protein would allow the PstSCAB transporter to be open for Pi transportation at all times, even under the high concentration of phosphate in eutrophic water bodies.

With the purpose of proving the effectiveness of our engineered E. coli in decreasing the concentration of inorganic phosphate (Pi) in the environment, 5-Bromo-4-chloro-3-indolyl phosphate coloration and malachite green coloration are used.

To ensure the effectiveness and practicality of our aforementioned experimental design, we conducted a preliminary experiment. 5-Bromo-4-chloro-3-indolyl phosphate (XP) is a chromogenic substrate that shows no color in its stable state. However, if XP is in contact with PhoA, PhoA will hydrolyze XP, thus severing phosphate ions (a phosphate monoester + water = 5,5′-dibromo-4,4′-dichloro-indigo + a phosphate ion), and the remaining chemical will appear to be blue. Once an increase in the concentration of Pi is detected, the expression of PhoA rises and turns the XP solution blue. To test the function of our bacteria engineered with AsPhoU, we placed it inside the XP chemical to determine the absorbance of Pi by observing the color changes.

Our positive control group is E. coli DH5α in a low phosphate medium, which turns the XP solution blue, reflecting low phosphate to induce PhoA expression. On the contrary, no blue color was observed in a high phosphate medium.

After the addition of E. coli DH5α (with AsPhoU) with 0.2 % arabinose at a high phosphate concentration, the XP solution turned blue, proving the expression of PhoA and, thus, the transportation of Pi into the bacterial cells.

Once molybdate (MoO₄⁻²) comes into contact with phosphate (PO₄⁻³), a complex containing phosphomolybdic acid would usually form. This complex interacts with malachite, forming a green chromogenic complex that remains in its most stable phase in an acidic environment. The color formation runs proportional to the increase of phosphate concentration. The shifts in color could determine the absorbance at 620 nm by a spectrophotometer. We would add the aforementioned chemical into our bacteria to interpret the effectiveness of our engineering method.

Our team constructed a standard curve that measures the absorbance at 620 nm versus the concentration of phosphate solution in the presence of E. coli of 0.1 O.D.

We aimed to test the optimal time of our engineered E. coli functioning under different conditions. All simulated high phosphate solutions are prepared with 2 mM of K₂HPO₄, 0.06% glucose, and MOPS buffer; all simulated low phosphate solutions are prepared with 0.1 mM of K₂HPO₄, 0.4% glucose, and MOPS buffer.

We incubated our engineered E. coli (cultured at 0.1 O.D.) under the same condition, added malachite and molybdate into our E. coli colonies, and detected the absorbance at 600 and 620 nm, respectively, reflecting cell density and inorganic phosphate (Pi) concentration at different time points. Using the standard curve above, we translated the detected data of absorbance at 620 nm into phosphate concentration in μM.

Experiments on the bacterial body containing AsPhoU in the presence or absence of arabinose were conducted for the level of phosphate absorption. The groups with arabinose induction clearly reported higher absorbed phosphate concentrations as AsPhoU is expressed. More phosphate absorption in presence of arabinose was observed compared to the absence of arabinose in AsPhoU. The maximal amount of Pi absorption was 3.1 x 10-6 μM per CFU (colony forming unit) for 1-hour culture, estimating 2.52 mM phosphate absorption per OD.

The full set experimental bar graph (1, 2, 3 hrs) indicating efficiency are illustrated below:

Concluding from the previous experiments, AsPhoU cell has proven its effectiveness in absorbing a higher amount of Pi in the environment. While acknowledging that, we simultaneously have to make sure that the absorbed Pi constructively fixates into inorganic polyphosphate (polyP) so that it remains inside the bacterial bodies. Henceforth our team utilized Sigma-Aldrich’s PolyP assay kit, which helped in quantifying the amount of PolyP in bacterial bodies.

PolyP reacts fully with the fluorescent dye provided by the assay kit, forming a detectable complex. The fluorescence of the created complex is then measured with a spectrophotometer at λ (excitation) = 415 nm and λ (emission) = 550 nm. The amount of fluorescence detected runs proportional to the concentration of PolyP in the sampled bacteria.

Our team constructed a standard curve with the diluted PolyP solution and our assay buffer. The experimental groups are listed below:

Owing to time constraints, we have not been able to test the ability of AsPhoU for intracellular PolyP accumulation. Regardless, we would conduct this experimental process in the future, with the hope of reiterating that our proposed solution successfully operates. Moreover, we would subclone oph and AsPhoU to pACYCDuet vector with p15A ori, compatible with PolyP sensor carrying pMB ori to generate our PolyP sensor cell in this project.

Jha, Ramesh K., et al. “A Microbial Sensor for Organophosphate Hydrolysis Exploiting an Engineered Specificity Switch in a Transcription Factor.” Nucleic Acids Research, vol. 44, no. 17, 2016, pp. 8490–500. Crossref, https://doi.org/10.1093/nar/gkw687.

Devine, Kevin M. “Activation of the PhoPR-Mediated Response to Phosphate Limitation Is Regulated by Wall Teichoic Acid Metabolism in Bacillus subtilis.” Frontiers in microbiology vol. 9 2678. 6 Nov. 2018, doi:10.3389/fmicb.2018.02678

Haldimann, A et al. “Use of New Methods for Construction of Tightly Regulated Arabinose and Rhamnose Promoter Fusions in Studies of the Escherichia coli Phosphate Regulon.” Journal of bacteriology vol. 180,5 (1998): 1277-86. doi:10.1128/JB.180.5.1277-1286.1998