Difference between revisions of "Part:BBa K3332067"

Latest revision as of 05:57, 2 October 2024

J23100-RBS-phnE1-RBS-phnE2

A composite part that can enrich glyphosate in cytoplasm.To construct a new part that can transport glyphosate to cytoplasm

Biology

Phn system is a gene cluster for organophosphorus transport and degradation in many microorganisms. Sinorhizobium meliloti 1021 use PhnE1 and PhnE2 to construct the permease protein of ABC transporter, which includes PhnE1, PhnE2, PhnC and PhnD proteins, this transporter can transport glyphosate to cytoplasm. The phnEE gene encodes permease protein which can transport glyphosate to cytoplasm.

Usage

We ligased the strong promoter and RBS（BBa_J23100+BBa_B0034）and the parts (phnE1、phnE2) on the expression vector pSB1C3 by standard assembly. Then the ligation mixture was transformed into E. coli DH5α & E. coli BL21(DE3), which enabled the E. coli to transport glyphosate to cytoplasm.

Characterization

1. Agarose Gel Electrophoresis： After receiving the synthesized DNA, restriction digestion was done to certify that the plasmid was correct, and the experimental results were shown in figure 1.

Fig.1 The result of plasmid cut with enzyme EcoR I and Pst I. Plasmid: pSB1C3

2. SDS-PAGE: The constructed plasmid was transformed into E. coli BL21 (DE3). Both of them were electrophoresed on a sodium dodecyl sulfate (SDS)-12% (wt/vol) polyacrylamide gel, followed by silver staining.

Fig.2 SDS-PAGE analysis of protein in lysate of E. coli BL21 (DE3) cells. Target bands can be seen at about 36.1 kDa and 55.4kDa.

3. HPLC: Verify that phnEE enhances the transport of glyphosate by the chassis bacteria by HPLC. The result is shown in Fig.3, phnEE enhance the transport of glyphosate by the chassis bacteria compared to the blank control obviously.

Fig.3 Relationship between elution peak area of glyphosate and culture time.

Contributed by WHHS-China 2024

Glyphosate Transport

Effective degradation of glyphosate requires its efficient transport into E. coli cells. We introduced the phnE1 and phnE2 genes from Sinorhizobium meliloti 1021, which encode phosphonate acid transporters. Studies have shown that Sinorhizobium meliloti 1021 can absorb 85% of glyphosate within less than an hour. By expressing the PhnE proteins, glyphosate is quickly transported across the cell membrane, enabling subsequent degradation.

Figure 1. Diagram showing glyphosate transport and genetic circuit.

Characterization

phnE1/E2 glyphosate engineering strain:

Objective and Methods

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.

Figure 2: Agarose gel electrophoresis of phnE1 and phnE2 nucleic acids after PCR amplification

Results and Conclusion

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.

Verification of Glyphosate Absorption Ability in phnE1/E2 Engineered Strain:=

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.

Figure 3: Glyphosate concentration in the culture medium of phnE1/E2 engineered strain after 3 hours, measured using ELISA.

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.

Testing the Time Curve of Glyphosate Absorption in the phnE1/E2 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.

Figure 4: Time course of glyphosate absorption by the phnE1/E2 engineered strain over 5 hours. Glyphosate concentration was measured hourly using ELISA detection, with significant reduction seen over time.

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.

Sequence and Features