The aim was to construct an engineered E. coli BL21 strain by synthesizing the codon-optimized phnE1 and phnE2 genes from Sinorhizobium meliloti 1021. These genes were co-expressed in the pSB1A3 vector. The recombinant plasmid was transformed into E. coli BL21 and cultured in LB medium containing 50 µg/mL ampicillin at 37°C to select for the engineered strain.

The recombinant plasmid containing the phnE1 and phnE2 genes was successfully constructed and transformed into E. coli BL21. The engineered strain grew in LB medium supplemented with ampicillin, confirming successful plasmid incorporation and selection. This strain is now ready for further testing related to glyphosate absorption.

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.

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.

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.

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.

The aim of this experiment was to construct an engineered E. coli BL21 strain expressing phnE1, phnE2, and phnJ genes. The genes phnE1 and phnE2 (from Sinorhizobium meliloti 1021) were already inserted into a recombinant plasmid. In this step, the phnJ gene from Enterobacter cloacae K7 was synthesized and cloned downstream of the phnE1/E2 sequence in the pSB1A3 vector, under the control of the J23100 promoter. The recombinant plasmid, containing all three genes, was then transformed into E. coli BL21. The engineered strain was selected and cultured in LB medium containing 50 µg/mL ampicillin at 37°C.

The recombinant plasmid containing the phnE1, phnE2, and phnJ genes was successfully constructed and transformed into E. coli BL21. The engineered strain grew in LB medium supplemented with ampicillin, indicating the successful incorporation of the plasmid. The agarose gel electrophoresis confirmed the correct insertion of the three target genes, validating the construction of the desired plasmid.

In conclusion, the phnE1/E2-phnJ engineered strain was successfully constructed, laying the foundation for subsequent experiments to evaluate its glyphosate degradation capability.

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.

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 objective of this experiment was to construct an engineered E. coli BL21 strain expressing the phnO gene, which encodes an enzyme involved in the degradation of aminomethylphosphonic acid (AMPA), a major byproduct of glyphosate breakdown. The phnO gene from Salmonella enterica LT2 was codon-optimized for expression in E. coli and cloned into the pET23b vector using the EcoRI and XhoI restriction sites. The recombinant plasmid was transformed into E. coli BL21, and the engineered strain was selected by growing in LB medium containing 50 µg/mL ampicillin at 37°C.

The recombinant plasmid containing the phnO gene was successfully constructed and transformed into E. coli BL21. The engineered strain grew robustly in LB medium containing ampicillin, indicating successful plasmid incorporation and expression of the gene. Gel electrophoresis confirmed the correct insertion of phnO into the vector.

In conclusion, the phnO engineered strain was successfully constructed, providing a basis for further studies on its role in AMPA degradation, which will be critical for advancing the glyphosate degradation pathway.

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.

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.

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.

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.

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.

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 objective of this experiment was to construct a cold-inducible reporter strain using a cold-inducible promoter to regulate the expression of the mRFP (monomeric red fluorescent protein) gene. This strain allows for the detection of gene expression under low-temperature conditions.

The cold-inducible promoter PcspAwas synthesized and cloned upstream of the mRFP gene in the pSB1A3 vector. The mRFP gene was codon-optimized for expression in E. coli, and restriction sites (EcoRI, XbaI, SpeI, PstI) were removed to comply with RFC#10 standards. The recombinant plasmid was transformed into E. coli BL21 cells. Positive clones were selected on LB agar plates containing 50 µg/mL ampicillin at 37°C and confirmed by sequencing.

The engineered strain was cultured in LB medium containing ampicillin to evaluate the induction of mRFP expression under different temperatures.

The recombinant plasmid containing the cold-inducible PcspA promoter and the mRFP gene was successfully constructed and transformed into E. coli BL21. Sequencing confirmed the correct insertion of the promoter and reporter gene into the pSB1A3 vector. The engineered strain exhibited normal growth in LB medium containing ampicillin, demonstrating successful plasmid incorporation and expression capability.

In conclusion, the construction of the cold-inducible reporter strain was successful, providing a functional system for studying temperature-dependent gene expression. This strain will be used in subsequent experiments to monitor mRFP expression under varying cold conditions.

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.

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.

The aim of this experiment was to construct a cold-inducible suicide system in E. coli using the cold-inducible PcspA promoter to control the expression of the mazF gene, which encodes a toxin that inhibits cellular growth by cleaving mRNA. This system is designed to activate cell death under low-temperature conditions, providing a potential biocontainment strategy.

The mazF gene, codon-optimized for E. coli, was cloned downstream of the PcspA promoter in the pSB1A3 vector. The PcspA-mazF construct was transformed into E. coli BL21 cells, and positive clones were selected on LB agar plates containing 50 µg/mL ampicillin at 37°C. The presence of the PcspA-mazF system was confirmed by sequencing.

The engineered strain was cultured under standard growth conditions (37°C), and subsequent experiments were planned to assess the activation of the suicide system at lower temperatures.

The recombinant plasmid containing the PcspA promoter and mazF toxin gene was successfully constructed and transformed into E. coli BL21. Sequencing confirmed the correct insertion of the mazF gene under the control of the cold-inducible promoter. The engineered strain grew normally at 37°C in LB medium containing ampicillin, confirming successful plasmid incorporation and maintenance of growth under non-inducing conditions.

In conclusion, the construction of the cold-inducible suicide system was successful, providing a functional system that can potentially trigger cell death in response to cold temperatures. This strain will be used in subsequent experiments to test the effectiveness of the suicide system at inducing cell death under low-temperature conditions.

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.

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.

All statistical analyses were performed using GraphPad Prism software. Data are presented as mean ± standard deviation (SD), with experiments conducted in triplicate to ensure reliability. For multiple group comparisons, one-way ANOVA followed by Tukey’s post-hoc test was applied. Comparisons between two groups were analyzed using a two-tailed Student’s t-test. A p-value of < 0.05 was considered statistically significant. These analyses verified that the observed differences, such as in growth inhibition and temperature-dependent gene expression, were statistically significant and not due to random variation, confirming the validity of the experimental results.

In conclusion, the successful construction of the phnE1/E2, phnE1/E2-phnJ, and phnO engineered strains demonstrated effective glyphosate and AMPA degradation, confirming the functional roles of these genes. The cold-inducible reporter and suicide systems were also validated, with the PcspA promoter effectively regulating gene expression and inducing cell death at low temperatures. These results highlight the potential of the engineered strains for environmental bioremediation and biosafety applications, offering innovative solutions for glyphosate degradation and biocontainment.

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