This project aimed to engineer E. coli strains capable of efficient glyphosate absorption, degradation, and biocontainment. The first cycle focused on constructing a strain expressing phnE1 and phnE2 genes to enhance glyphosate uptake. In the second cycle, the phnJ gene was added to improve degradation, although limited success led to addressing AMPA byproduct degradation in the third cycle through the introduction of phnO. Finally, to address biosafety concerns, a cold-inducible suicide system using the PcspA promoter and mazF toxin was developed to ensure controlled bacterial growth in the environment.

The goal of this cycle was to construct an E. coli BL21 strain that could absorb glyphosate more efficiently by expressing the phnE1 and phnE2 genes from Sinorhizobium meliloti 1021. These genes are responsible for encoding enzymes that facilitate the transport of glyphosate into the bacterial cell. By introducing both genes, we aimed to improve the uptake of glyphosate, which is a crucial first step before its degradation.

Key considerations in the design:

phnE1 and phnE2 genes were selected for their known role in glyphosate transport.

The genes were codon-optimized for efficient expression in E. coli BL21.

The pSB1A3 plasmid was chosen as the vector, which includes an ampicillin resistance gene for selection purposes.

The construction process involved several steps to ensure the successful expression of phnE1 and phnE2 in E. coli:

Gene Synthesis and Optimization: The phnE1 and phnE2 genes were synthesized with codon optimization to enhance expression in E. coli. This step is crucial to ensure that the bacterial system can effectively translate these genes into functional proteins.

Cloning into Vector: Both genes were cloned into the pSB1A3 vector. This vector was selected due to its compatibility with E. coli and its use in iGEM standards for modular cloning. The cloning involved using restriction enzyme digestion followed by ligation to insert the genes into the plasmid.

Transformation: The recombinant plasmid was transformed into E. coli BL21. The transformed cells were grown on LB agar plates containing ampicillin (50 µg/mL) to select for successful transformants. Only cells that successfully incorporated the plasmid would grow in the presence of ampicillin.

Verification of Construction: The successful integration of the phnE1 and phnE2 genes was confirmed through PCR and agarose gel electrophoresis, verifying that the genes had been correctly inserted into the plasmid.

Once the engineered strain expressing phnE1/E2 was constructed, two key tests were performed to evaluate the strain’s ability to absorb glyphosate.

Objective and Methods

The purpose of this experiment was to evaluate the glyphosate absorption capacity of the engineered E. coli strain expressing phnE1 and phnE2. The engineered strain was first cultured overnight in LB medium containing 100 µg/mL ampicillin. The next day, the culture was diluted 1:100 into LB medium supplemented with 80 mg/L glyphosate and 50 µg/mL ampicillin and incubated for 3 hours. After incubation, the cultures were centrifuged, and the supernatant was collected for glyphosate concentration analysis using an ELISA detection kit. Absorbance was measured at 450 nm to determine the glyphosate levels.

Results and Conclusion

The engineered E. coli strain expressing phnE1 and phnE2 exhibited a significant reduction in glyphosate concentration in the medium after 3 hours of incubation. This indicates that the strain was able to absorb glyphosate efficiently. The results confirm the functional expression of the phnE1 and phnE2 genes, enhancing the glyphosate uptake ability of the engineered strain.

Objective and Methods

The objective of this experiment was to evaluate the glyphosate absorption capacity of the engineered E. coli strain expressing phnE1 and phnE2 over a 5-hour period. The strain was first cultured overnight and then diluted 1:100 into LB medium containing 80 mg/L glyphosate and 50 µg/mL ampicillin. The cultures were incubated at 37°C with shaking, and 1 mL samples were taken at hourly intervals for 5 hours. After centrifugation, the supernatant was collected, and glyphosate concentration was analyzed using an ELISA detection kit. Absorbance at 450 nm was measured, and the glyphosate concentration was calculated based on a standard curve.

Results and Conclusion

The time-course analysis showed a steady decrease in glyphosate concentration over the 5-hour period. After 1 hour, there was a substantial reduction in glyphosate levels, indicating rapid uptake by the engineered strain. Over the next four hours, the absorption continued, though at a slightly slower rate. By the 5th hour, glyphosate concentration in the medium had decreased by over 60%, confirming the strain’s sustained absorption capability.

In conclusion, the phnE1/E2 engineered strain demonstrated effective and continuous absorption of glyphosate over the 5-hour testing period, with the highest rate of uptake occurring in the first hour. This time curve highlights the strain’s potential for extended applications in glyphosate removal.

From the results of the tests, we confirmed that the engineered phnE1/E2 strain was capable of absorbing glyphosate effectively. However, this is only the first step in the overall process of glyphosate degradation. Once absorbed, the glyphosate needs to be broken down within the cell to mitigate its environmental impact.

Following the successful absorption of glyphosate in the first cycle, the focus of this cycle was to enhance the degradation of glyphosate after it was absorbed by the phnE1/E2 engineered strain. The degradation of glyphosate requires a series of enzymatic steps, and we hypothesized that the expression of the phnJ gene could accelerate this process.

phnJ encodes a phosphonatase enzyme responsible for catalyzing the breakdown of glyphosate into less harmful byproducts. The goal was to co-express phnJ along with phnE1/E2 to form a more efficient system capable of both absorbing and degrading glyphosate.

The J23100 promoter was selected to drive the expression of phnJ, ensuring constitutive expression of the enzyme in the engineered strain. The plasmid vector pSB1A3 was again used for consistency with previous cycles, and for maintaining ampicillin resistance as a selection marker.

The construction process in this cycle involved cloning the phnJ gene into the plasmid already containing the phnE1 and phnE2 genes, and ensuring that all three genes were properly expressed in E. coli BL21.

Gene Synthesis and Insertion: The phnJ gene from Enterobacter cloacae K7 was synthesized with codon optimization for expression in E. coli. It was then inserted downstream of the phnE1/E2 gene sequence in the pSB1A3 plasmid using restriction enzyme digestion and ligation.

Plasmid Construction and Transformation: The recombinant plasmid, now containing phnE1/E2 and phnJ, was transformed into E. coli BL21 via heat shock. The cells were grown on LB agar plates containing ampicillin to select for transformants carrying the plasmid.

Verification: PCR amplification was used to verify the correct insertion of phnJ. Gel electrophoresis confirmed the presence of the desired gene fragments, ensuring that the plasmid had been successfully constructed.

Objective and Methods

The goal of this experiment was to evaluate the glyphosate degradation capability of the phnE1/E2-phnJ engineered E. coli strain. The strain was first cultured overnight, then diluted 1:100 into LB medium containing 80 mg/L glyphosate and 50 µg/mL ampicillin. The cultures were incubated at 37°C with shaking for 3 hours. Samples were collected at 0 hours and 3 hours, centrifuged, and the supernatant was analyzed using an ELISA detection kit. Absorbance was measured at 450 nm to determine the glyphosate concentration, which was then calculated using a standard curve.

Results and Conclusion

The results showed that both the phnE1/E2-phnJ and phnE1/E2 engineered strains degraded glyphosate over the 3-hour incubation period, but there was no significant difference between the two strains in terms of degradation efficiency. Despite the additional expression of the phnJ gene in the phnE1/E2-phnJ strain, glyphosate concentration in the medium decreased similarly in both strains.

Upon further analysis and discussion, it was hypothesized that the rate-limiting step in glyphosate degradation may be the transport of glyphosate into the cells, rather than the intracellular enzymatic degradation. This could explain why the extra expression of phnJ did not result in a significant improvement in degradation efficiency. Future experiments may focus on optimizing the transport mechanisms to enhance the overall degradation process.

In conclusion, although the phnE1/E2-phnJ strain demonstrated effective glyphosate degradation, the results suggest that transport, rather than enzymatic breakdown, may be the bottleneck in the degradation pathway. Further work is needed to address this limitation.

The results from this cycle indicated that despite the successful introduction and expression of the phnJ gene, no significant improvement in glyphosate degradation was observed compared to the phnE1/E2 strain. This suggested that the degradation bottleneck might not be the enzymatic breakdown within the cell but rather the presence of AMPA, a toxic byproduct of glyphosate degradation, which remained in the medium. Therefore, the focus for the next cycle shifted towards degrading AMPA, leading to the design of the phnO engineered strain.

After observing AMPA accumulation during glyphosate degradation in the second cycle, the third cycle aimed to degrade AMPA, a toxic byproduct of glyphosate breakdown. The strategy involved the expression of the phnO gene, which encodes an enzyme responsible for converting AMPA into a less harmful product via N-acetylation.

phnO from Salmonella enterica LT2 was selected for this task. The enzyme it encodes, AMPA N-acetyltransferase, catalyzes the acetylation of AMPA, rendering it non-toxic.

The pET23b plasmid was chosen for this cycle to provide robust expression of phnO under control of the T7 promoter. This vector also contains an ampicillin resistance gene, ensuring consistency in selection with previous cycles.

To construct the engineered strain capable of degrading AMPA, the following steps were carried out:

Gene Synthesis and Cloning: The phnO gene was codon-optimized for expression in E. coli and synthesized. It was cloned into the pET23b plasmid using EcoRI and XhoI restriction sites to ensure correct orientation and expression.

Transformation: The recombinant phnO plasmid was transformed into E. coli BL21 using heat shock transformation. Successful transformants were selected on LB agar plates containing ampicillin (50 µg/mL).

Verification: To confirm successful plasmid construction, colony PCR was conducted followed by gel electrophoresis to verify the presence of phnO in the plasmid. Sequencing was also performed to ensure the correct insertion of the gene.

With the engineered phnO strain constructed, several tests were performed to assess its ability to degrade AMPA and the efficiency of the enzyme.

Objective and Methods

The purpose of this experiment was to assess the ability of the phnO engineered strain to degrade aminomethylphosphonic acid (AMPA), a toxic byproduct of glyphosate breakdown. Accumulation of AMPA in the environment poses potential ecological and health risks, making its degradation crucial for effective bioremediation. The phnO gene, encoding an AMPA N-acetyltransferase, was expressed in E. coli BL21 to enable the degradation of this harmful compound.

The engineered strain was cultured overnight in LB medium containing 50 µg/mL ampicillin at 37°C. The next day, the culture was diluted 1:100 into fresh LB medium and grown until an OD600 of 0.6 was reached. Cells were collected by centrifugation, resuspended in pre-cooled Tris-HCl buffer, and lysed via sonication to obtain a crude enzyme extract. The reaction mixture, containing 1 mM AMPA, acetyl-CoA, and magnesium chloride, was incubated with the crude extract at 37°C for 3 hours. The degradation of AMPA was monitored by measuring CoA production using a DTDP-based colorimetric assay at 324 nm.

Results and Conclusion

The phnO engineered strain demonstrated a significant capacity to degrade AMPA, as evidenced by the increase in CoA production in the reaction mixture. The absorbance at 324 nm confirmed that the phnO enzyme successfully catalyzed the acetylation of AMPA, leading to its degradation. The reduction of this toxic byproduct is a critical step in mitigating the environmental and health hazards associated with glyphosate use.

In conclusion, the phnO strain efficiently degraded the toxic compound AMPA, highlighting the strain’s potential for comprehensive glyphosate degradation. By breaking down AMPA, this engineered strain provides a valuable tool for reducing the harmful effects of glyphosate byproducts in bioremediation efforts.

Objective and Methods

The objective of this experiment was to evaluate the time-dependent degradation of aminomethylphosphonic acid (AMPA) by the phnO engineered strain. AMPA, a toxic byproduct of glyphosate degradation, poses environmental and health risks, making its efficient degradation critical. The engineered strain expressing the phnO gene was tested for its ability to degrade AMPA over a 5-hour period.

The strain was cultured overnight in LB medium containing 50 µg/mL ampicillin. The following day, the culture was diluted 1:100 into fresh LB medium and incubated at 37°C until an OD600 of 0.6 was reached. After centrifugation, the cells were resuspended in pre-cooled Tris-HCl buffer and lysed by sonication to prepare a crude enzyme extract.

The reaction mixture containing 1 mM AMPA, acetyl-CoA, and magnesium chloride was incubated with the crude enzyme extract at 37°C. Samples were taken at hourly intervals for 5 hours, and CoA production was measured using the DTDP assay by recording the absorbance at 324 nm. The production of CoA indicated the degradation of AMPA by the phnO enzyme over time.

Results and Conclusion

The time-course analysis revealed a steady increase in CoA production over the 5-hour period, indicating continuous degradation of AMPA by the phnO enzyme. The rate of degradation was highest during the first two hours, after which it slowed slightly but remained consistent throughout the experiment. By the 5th hour, a significant reduction in AMPA levels was observed, confirming the enzyme’s sustained activity.

In conclusion, the phnO engineered strain efficiently degraded AMPA over time, with the most rapid degradation occurring in the initial stages. The ability of the strain to maintain enzyme activity over several hours highlights its potential for use in bioremediation processes aimed at mitigating the harmful effects of glyphosate byproducts, such as AMPA.

Objective and Methods

The objective of this experiment was to determine the kinetic parameters of the phnO enzyme expressed in E. coli BL21. The enzyme catalyzes the degradation of aminomethylphosphonic acid (AMPA), a toxic byproduct of glyphosate degradation. Understanding the enzyme’s kinetics is crucial for optimizing its application in bioremediation.

The engineered strain was cultured overnight in LB medium containing 50 µg/mL ampicillin at 37°C. The next day, the culture was diluted 1:100 into fresh LB medium and grown until an OD600 of 0.6. Cells were harvested by centrifugation, resuspended in pre-cooled Tris-HCl buffer, and lysed via sonication to prepare a crude enzyme extract.

To determine the enzyme kinetics, reaction mixtures were prepared with varying concentrations of AMPA and incubated with the crude enzyme extract at 37°C for 3 hours. CoA production was measured using the DTDP assay, and absorbance was recorded at 324 nm. The Michaelis constant (Km) and maximum reaction velocity (Vmax) were calculated using nonlinear regression analysis of the data, plotted as a Michaelis-Menten curve.

Results and Conclusion

The Michaelis-Menten kinetic analysis revealed that the phnO enzyme followed saturation kinetics with increasing concentrations of AMPA. The calculated Km value was 0.5958 mM, indicating a moderate affinity for AMPA. The Vmax was 0.1828 µmol/min/mg protein, reflecting the maximum reaction velocity achieved when the enzyme was saturated with AMPA.

These kinetic parameters demonstrate that the phnO enzyme is effective at catalyzing AMPA degradation at moderate substrate concentrations. The relatively low Km value suggests that the enzyme is well-suited for bioremediation purposes where the environmental concentration of AMPA is likely to be low to moderate.

In conclusion, the kinetic analysis of the phnO enzyme confirmed its ability to efficiently degrade the toxic byproduct AMPA. The specific values of Km and Vmax provide valuable insights for optimizing its application in environmental detoxification strategies.

The results from this cycle demonstrated that the engineered phnO strain was able to effectively degrade AMPA, reducing the accumulation of this toxic byproduct. However, while the strain successfully handled AMPA degradation, there were concerns regarding the potential uncontrolled spread of the engineered bacteria in the environment, raising biosafety issues. This realization led to the development of a biocontainment system for the next cycle, focused on creating a cold-inducible suicide system to prevent the bacteria from proliferating outside of controlled conditions.

Following the success in degrading AMPA in the third cycle, the next focus was on biosafety. To prevent the potential uncontrolled spread of the engineered bacteria in natural environments, a cold-inducible suicide system was designed. This system would trigger cell death when the bacteria are exposed to lower temperatures, ensuring containment outside of laboratory conditions.

The PcspA promoter, which is cold-inducible, was selected to control the expression of the mazF gene. mazF encodes a toxin that cleaves cellular mRNA, leading to cell death. The idea was that at temperatures below 16°C, PcspA would activate mazF expression, inducing the suicide mechanism.

The plasmid pSB1A3 was chosen to maintain consistency with previous cycles, with ampicillin resistance used for selection.

The construction of the cold-inducible suicide system involved cloning the cold-sensitive regulatory elements and ensuring functional expression of the mazF toxin.

Gene Cloning: The mazF gene was codon-optimized for expression in E. coli. It was cloned downstream of the PcspA promoter in the pSB1A3 vector, ensuring that its expression would only occur under cold temperatures.

Transformation: The recombinant plasmid was transformed into E. coli BL21 via heat shock, and transformants were selected on LB agar plates containing ampicillin.

Verification: Colony PCR was performed to verify the correct insertion of the PcspA-mazF construct, followed by sequencing to ensure the accurate assembly of the plasmid.

With the cold-inducible suicide system in place, several tests were carried out to verify the functionality of the PcspA-mazF system in response to different temperatures.

Objective and Methods

The objective of this experiment was to test the cold-inducible reporter strain constructed with the PcspA promoter driving the expression of mRFP (monomeric red fluorescent protein). The goal was to evaluate the induction of mRFP expression at different temperatures and determine the temperature sensitivity of the cold-inducible system.

The overnight culture of the engineered strain was diluted 1:100 into fresh LB medium containing 50 µg/mL ampicillin. The cultures were then incubated at different temperatures (16°Cand 37°C) with shaking at 180 rpm for 12 hours. After incubation, 200 µL samples were taken from each culture, and fluorescence intensity (excitation at 584 nm and emission at 607 nm) was measured using a microplate reader. Optical density at 600 nm (OD600) was also measured to normalize the fluorescence values. The normalized fluorescence intensity (Fluorescence/OD600) was calculated to compare mRFP expression levels across different temperature conditions.

Results and Conclusion

The results showed a significant increase in mRFP expression at lower temperatures, with the highest fluorescence observed at 16°C. As the temperature increased, the fluorescence intensity decreased, with minimal expression at 37°C. This indicates that the PcspA promoter is highly sensitive to cold temperatures, and mRFP expression is strongly induced under cold-inducible conditions.

In conclusion, the cold-inducible reporter strain successfully demonstrated temperature-dependent expression of mRFP, with optimal induction occurring at 16°C. This strain can be effectively used to study cold-inducible gene expression systems and could be applied to temperature-controlled biotechnological processes.

Objective and Methods

The purpose of this experiment was to test the functionality of the cold-inducible suicide system in E. coli, which utilizes the PcspA promoter to control the expression of the mazF toxin gene. The mazF gene induces cell death by cleaving mRNA, and this system is designed to activate at lower temperatures, offering a temperature-sensitive biocontainment strategy.

The engineered strain was cultured overnight in LB medium containing 50 µg/mL ampicillin at 37°C. The following day, 100 µL of the culture was inoculated into 5 mL of fresh LB medium containing ampicillin and incubated at either 16°C or 37°C with shaking at 180 rpm for 12 hours. After incubation, 200 µL samples were taken from each condition, and cell growth was measured by determining the optical density at 600 nm (OD600). Cell viability at low temperatures was compared with that of control strains and normal growth conditions at 37°C.

Results and Conclusion

The results showed that at 37°C, the engineered strain grew normally, with no signs of mazF-induced cell death. However, when incubated at 16°C, the growth of the engineered strain was significantly inhibited, with a drastic reduction in OD600, indicating the activation of the suicide system. The control strain without the mazF gene showed normal growth at both 16°C and 37°C, confirming that the observed growth inhibition was due to the cold-inducible expression of mazF.

In conclusion, the cold-inducible suicide system effectively triggered cell death at 16°C, validating the functionality of the PcspA-regulated mazF system. This system provides a potential safety mechanism for biocontainment, ensuring that the engineered bacteria can be selectively eliminated under specific environmental conditions.

The cold-inducible suicide system proved effective in triggering cell death at low temperatures, providing a reliable biocontainment mechanism. This system ensures that the engineered bacteria can be safely controlled in the environment, as the suicide gene is activated when the bacteria leave the controlled, warmer laboratory environment. With the cold-inducible PcspA promoter successfully regulating gene expression, the system is a valuable tool for biosafety, reducing risks associated with the release of genetically modified organisms into the wild.

The project successfully developed engineered E. coli strains that absorbed and degraded glyphosate and AMPA efficiently while implementing a cold-inducible suicide system for environmental biosafety. While the introduction of the phnJ gene did not enhance degradation, the addition of phnO effectively degraded AMPA. The cold-inducible suicide system provided an essential biocontainment mechanism, making the engineered bacteria safer for environmental applications in glyphosate bioremediation.