Overview
Heavy metals in water pose serious health risks. Our project focused on removing heavy metal ions from water by engineering E. coli to express rice metallothionein (OsMTI-1b) for metal binding and a urease gene cluster from Sporosarcina pasteurii for Microbiologically Induced Calcite Precipitation (MICP). In tests, the strain expressing both genes demonstrated the best overall performance, with OsMT1 excelling at lower concentrations and the urease gene cluster being more efficient at higher concentrations, achieving maximum removal rates of 85.78% for cadmium and 98.98% for lead. Our engineered DH5α strain is scalable, eco-friendly, and ideal for use in industrial wastewater treatment plants.
The following are our contributions:
Introducing an Efficient Cell Membrane Display System for E. coli
The Non sequence is an artificially designed signal-like sequence that directs newly synthesized proteins to the cell membrane via a non-classical secretion pathway. The OmpA (outer membrane protein A) fragment (amino acids 46-159) spans part of the extracellular domain in E. coli, helping display proteins on the cell membrane. This system combines the Non sequence with the extracellular fragment of OmpA to display proteins on the E. coli surface. This Non-OmpA system (https://parts.igem.org/Part:BBa_K5205003), introduced to the iGEM community by us, offers higher efficiency than the traditional Lpp-OmpA system.
Developed E. coli Strains that Efficiently and Stably Induce MICP
We developed E. coli strains that efficiently and stably induce MICP by introducing the complete urease gene cluster from S. pasteurii into E. coli DH5α. This was verified by culturing the strains in LB media containing CaCl₂ and urea. We believe that our MICP-inducing E. coli can assist future iGEM teams interested in topics including bioremediation, biocementation, soil stabilization, and erosion protection.
Figure 1. Overnight culture of engineered E. coli in LB-urea-calcium media. A. No MICP; B. E. coli DH5α harboring pET28a-Ure induced MICP. The dark grains were calcium carbonate precipitates.
Long Range PCR Troubleshooting
To assemble the pET28a-OsMT1-Ure recombinant vector, we planned to linearize the partial pET28a-OsMT1 and pET28a-Ure using PCR. With ordinary PCR setup, we managed to get the partial pET28a-OsMT1 (4277 bp) but failed to get partial pET28a-Ure (6956 bp), probably due to reduced PCR efficiency when amplifying a long fragment. Adjusting the template amount was a good try but did not help. After several failures, we tried touchdown PCR from 65°C to 50°C, and the PCR products showed clear bands. This troubleshooting approach can help future teams with long-range PCR. See Engineering Success https://2024.igem.wiki/hangzhou-sdg/engineering for more details.
Troubleshooting Non-Functionality in E. coli BL21
In our project, we introduced MICP-inducing genes into E. coli DH5α and BL21, but only observed functionality in DH5α. SDS-PAGE showed high protein expression in BL21, likely due to its excessive expression rate, which overwhelmed the cell’s folding machinery, causing misfolded proteins in inclusion bodies. In contrast, DH5α's lower expression levels likely allowed proper protein folding. Although adjusting culture conditions, such as lowering the temperature, might mitigate the issue of BL21, we chose DH5α for its stability. Our troubleshooting offers future iGEM teams insights on possible issues and solutions when facing non-functionality in engineered E. coli BL21. See Engineering Success https://2024.igem.wiki/hangzhou-sdg/engineering for more details.
Clarified Regulations of GMOs in China
The approval process of GMOs in China involves three trial stages: intermediate test, environmental release test, and production test, as outlined in the Biosecurity Law and the 2016 Measures for the Safety Evaluation of Agricultural GMOs. During each stage, factors like genetic stability, toxicity, spread, and non-target impacts are assessed. After completing trials, submission of the Application for the Safety Evaluation of Agricultural GMOs to the relevant authorities is required, and if approved, the Ministry of Agriculture and Rural Affairs will issue a safety certificate. We hope future iGEM teams can gain insights into regulations and adjust their projects accordingly. See Human Practices https://2024.igem.wiki/hangzhou-sdg/human-practices for more details.