Retrieve literature from Web of Science, categorize it based on impact factor and content, select representative biosensor models, and analyze their design principles and practical performance.

The experimental group was divided into three study teams, each consisting of two individuals. They entered the laboratory to learn experimental procedures, with each session lasting one hour. The training focused on molecular biology techniques.

Rapidly extracted genomic DNA from Cupriavidus metallidurans CML2 using microwave heating, designed primers, and amplified the gene encoding the ArsR protein from CML2 genomic DNA via polymerase chain reaction (PCR). Subsequently, the PCR product was cloned into the pET-46a expression vector using DNA recombination techniques and sent for sequencing.

Construction of recombinant pET46a-ArsR-sfGFP-V0: Genomic DNA from Cupriavidus metallidurans CML2 was extracted using the microwave method. PCR amplification of the ArsR protein gene was performed using forward primer and and reverse primer. The PCR product was then ligated into the pET-46a vector, which had been pre-cloned with sfGFP, using DNA recombination techniques.

Identify the promoters for ArsR and sfGFP through literature review, then design forward and reverse primers accordingly. Clone these into the pET46a-ArsR-sfGFP-V0 recombinant vector via homologous recombination.

E. coli was inoculated at 1:50 into 5 mL LB medium and cultured overnight. After induction with varying concentrations of NaAsO₂, the culture was incubated at 37°C for 6 hours. One milliliter of the culture was then centrifuged at 12,000 rpm for 1 minute, and the supernatant was discarded. The pellet was resuspended in 1 mL of PBS. A 200 μL aliquot was transferred to a black-bottomed 96-well plate.

Using designed forward and reverse primers, construct the truncated construct pET46a-ParsOC2-sfGFP (deleting PlacV-ArsR) via reverse PCR using pET-46a-PlacV-ArsR-ParsOC2-sfGFP-V1 as template; Based on this truncated construct, primers were designed to insert the ArsR protein-encoding gene into the ParsOC2 promoter via homologous recombination cloning. After successfully transforming the V1 plasmid into E. coli DH5α, performance testing was conducted.

Based on the recombinant pET-46a-ParsOC2-sfGFP (deleting PlacV-ArsR), primers were designed to amplify the sequence encoding the ArsR protein, its promoter, and the ribosomal binding site designated as PlacV-RBS (arsR). Using homologous recombination cloning, this sequence was inserted upstream of the ParsOC2 promoter. Following successful transformation of the V1 plasmid into E. coli DH5α, performance testing was conducted.

Construct the recombinant vector pET-46a-PJ100-ArsR-ParsOC2-sfGFP-V4 and conduct preliminary performance testing of the biosensor-V4: First, the target promoter sequence PJ100 was obtained from the online website Promoters/Catalog - parts.igem.org. Using reverse PCR, the PlacV element in the recombinant vector pET46a-PlacV-ArsR-ParsOC2-sfGFP-V3 was replaced. The resulting V1 plasmid was successfully transformed into E. coli DH5α for performance testing.

Construction of Dose-Response Curves for V3 and V4 Sensors: Attempts were made to construct dose-response curves for all available data, with subsequent discussions of the results. The outcomes were unsatisfactory.

Optimization Strategy: Isolate the natural promoter ParsCML2 from the rice endophytic bacterium Cupriavidus metallidurans CML2, and clarify its co-evolutionary characteristics with the homologous ArsR protein (enhanced adaptability). Retain the core architecture of "PJ100 driving ArsR," replacing only the ParsOC2 promoter in the original vector pET-46a-PJ100-ArsR-ParsOC2-sfGFP with ParsCML2. The target is to construct the recombinant plasmid pET-46a-PJ100-ArsR-ParsCML2-sfGFP-V4.

Optimization Strategy: Isolate the natural promoter ParsCML2 from the rice endophytic bacterium Cupriavidus metallidurans CML2, and clarify its co-evolutionary characteristics with the homologous ArsR protein (enhanced adaptability). Retain the core architecture of "PJ100 driving ArsR," replacing only the ParsOC2 promoter in the original vector pET-46a-PJ100-ArsR-ParsOC2-sfGFP with ParsCML2. The target is to construct the recombinant plasmid pET-46a-PJ100-ArsR-ParsCML2-sfGFP-V4.

Mutagenesis Method (ParsCML2 Promoter Engineering): Employ error-prone PCR to introduce random mutations into the ParsCML2 promoter, including base substitutions, insertions, or deletions. Focus on the core functional regions of the ParsCML2 promoter (-35 region, -10 region). Establish a library size of 1000+ clones to ensure sufficient sequence diversity coverage.

Determine the self-assembly component and vector construction strategy: Select split-GFP fragments (GFP1-10, GFP11) and establish the "dual-signal dependency" logic (fluorescence is produced only when both "arsenic-induced ArsR repression release" and "GFP fragment self-assembly" occur simultaneously). Using pET46a-PlacV-ArsR-ParsOC2 as the base vector, clone GFP1-10 and GFP11 downstream of the ParsOC2 promoter to ensure synchronous expression of both fragments. Determine the induction timing as bacterial culture reaching OD600=0.6 (logarithmic phase).

Synthesize the GFP1-10 and GFP11 gene sequences and design specific primers containing homologous arms (matching the downstream sequence of the ParsOC2 vector). Amplify the GFP1-10 and GFP11 fragments via PCR, then digest the pET46a-PlacV-ArsR-ParsOC2 vector with restriction enzymes. Subsequently, the amplified GFP fragments were ligated to the digested vector via homologous recombination. The ligation products were transformed into E. coli competent cells and spread onto LB solid medium containing 50 μg/mL carbenicillin. The plates were incubated at 37°C overnight. Finally, single colonies were picked for colony PCR screening. Positive colonies were expanded, and plasmids were extracted. Sequencing validated the insertion position and sequence accuracy of the GFP fragment, confirming the successful construction of the "ArsR-ParsOC2-GFP fragment" self-assembling circuit.