Project Summary (July 1 – September 1)
From July 1st to September 1st, our iGEM team conducted a comprehensive set of synthetic biology experiments aimed at engineering E. coli for the inducible biosynthesis of neohesperidin and the integration of safety mechanisms. The primary achievements include:
- Construction of Neohesperidin-Producing Strains: We successfully synthesized and codon-optimized key genes—VvRHM, UGT73B2, Cm1,2RhaT, ThF3'H, CPR, F4′OMT—and cloned them into E. coli BL21 using the pET28a vector. Multicistronic operons were assembled for efficient expression.
Validation of New Synthetic Pathways:
- The neohesperidin synthesis pathway was induced with IPTG and tested at different temperatures, with 30°C yielding the highest output (~4.2 mg/mL).
- A parallel naringenin-to-hesperetin conversion pathway was tested using naringenin as the precursor, reaching ~11.9 mg/mL hesperetin output.
- Development of pH-Inducible Production Systems: Using the PcadC promoter, we implemented a low-pH inducible expression system for targeted drug production within tumor microenvironments. Reporter assays using mRFP confirmed functional pH sensitivity.
Establishment of Kill Switches: Two orthogonal biosafety modules were constructed:
- Arabinose-inducible suicide system using MAZF, verified to effectively kill bacteria in 0.2% arabinose.
- Cold-shock inducible system using the pCspA promoter to express MAZF under low temperatures.
Final Integrated Strain: An engineered probiotic (
E. coli Nissle 1917) was developed, capable of:
- Low-pH-induced neohesperidin biosynthesis.
- Arabinose- or low-temperature-triggered suicide for containment.
Based on the information you provided, here is:
Project Summary (July 1 – September 1)
From July 1st to September 1st, our iGEM team conducted a comprehensive set of synthetic biology experiments aimed at engineering E. coli for the inducible biosynthesis of neohesperidin and the integration of safety mechanisms. The primary achievements include:
- Construction of Neohesperidin-Producing Strains: We successfully synthesized and codon-optimized key genes—VvRHM, UGT73B2, Cm1,2RhaT, ThF3'H, CPR, F4′OMT—and cloned them into E. coli BL21 using the pET28a vector. Multicistronic operons were assembled for efficient expression.
Validation of New Synthetic Pathways:
- The neohesperidin synthesis pathway was induced with IPTG and tested at different temperatures, with 30°C yielding the highest output (~4.2 mg/mL).
- A parallel naringenin-to-hesperetin conversion pathway was tested using naringenin as the precursor, reaching ~11.9 mg/mL hesperetin output.
- Development of pH-Inducible Production Systems: Using the PcadC promoter, we implemented a low-pH inducible expression system for targeted drug production within tumor microenvironments. Reporter assays using mRFP confirmed functional pH sensitivity.
Establishment of Kill Switches: Two orthogonal biosafety modules were constructed:
- Arabinose-inducible suicide system using MAZF, verified to effectively kill bacteria in 0.2% arabinose.
- Cold-shock inducible system using the pCspA promoter to express MAZF under low temperatures.
Final Integrated Strain: An engineered probiotic (
E. coli Nissle 1917) was developed, capable of:
- Low-pH-induced neohesperidin biosynthesis.
- Arabinose- or low-temperature-triggered suicide for containment.
Daily Experimental Plan Summary (July 1 – September 1)
| Date |
Activity |
| July 1–3 |
Gene synthesis and plasmid receipt, codon optimization verification, primer design |
| July 4–8 |
PCR amplification of all synthetic genes, gel verification, and plasmid digestion |
| July 9–15 |
Ligation into pET28a vectors, transformation into E. coli BL21, colony PCR validation |
| July 16–20 |
Co-expression operon construction for both pathways (UGT73B2, VvRHM, etc.), transformation into BL21 |
| July 21–26 |
Test expression using IPTG induction (16°C, 25°C), SDS-PAGE verification of protein expression |
| July 27–31 |
Fermentation with hesperetin/naringenin substrate, initial product detection by HPLC |
| August 1–5 |
Optimization of fermentation temperature (25°C, 30°C, 37°C), identify 30°C as best yield |
| August 6–10 |
Construction and transformation of pH-inducible reporter system (PcadC-mRFP) |
| August 11–15 |
Characterization of inducible promoter at pH 5.8 vs pH 7.3, normalized fluorescence measurements |
| August 16–20 |
Integration of pH-inducible neohesperidin pathway into BL21, test production under pH 5.8 |
| August 21–25 |
Design and assembly of suicide circuits: pBAD-MAZF and pCspA-MAZF |
| August 26–28 |
Transformation of kill switch circuits into BL21, growth curve analysis, induction tests with arabinose/low temp |
| August 29–30 |
Final strain assembly: integration into E. coli Nissle 1917, verification of all modules |
| September 1 |
Final fermentation under induced conditions, record neohesperidin output, prepare for characterization and submission |
