[P008] Poster

1:00 PM - 1:50 PM JST(4:00 AM - 4:50 AM UTC)

[P04-504] Development of a high-sensitivity arsenic sensor through protein engineering.

○takahiro suzuki suzuki¹, Ryo Yamaguchi Yamaguchi¹, Katsumasa Kamiya Kamiya², Shigeko Kawai-Noma Kawai-Noma¹ (1. Dept. of Applied Chem. & Biotech., Grad. Sch. Eng., Chiba Univ. (Japan), 2. Center for Basic Edu. & Integ. Learn., Kanagawa Inst. Tech (Japan))

Keywords:

protein engineering,whole cell biosensor,transcriptional repressor,arsenite,cysteine

Purpose: Arsenic in groundwater is a severe health hazard, and developing regions urgently need inexpensive detection methods. Recent microbial fluorescence sensors use the repressor ArsR. Repressors bind regulatory DNA to block gene expression. ArsR binds the arsO sequence, but in the presence of arsenic, its cysteine (Cys) residues bind the metal, causing structural changes and dissociation from arsO. Placing the sfgfp fluorescent gene downstream of arsO allows arsenic detection via fluorescence. Meeting the WHO standard of 10 ppb requires higher sensitivity. This study focused on the tetracycline-responsive repressor TetR. While TetR does not naturally respond to arsenic, its structural and conformational change mechanisms are known. We redesigned TetR to release gene expression via structural changes induced by arsenic binding by introducing mutations like Cys at specific positions. This elucidates arsenic responsiveness principles to create a novel, highly sensitive sensor.MethodsSelection of Mutation Sites in TetR: Based on TetR structural data, we identified sites where conformational changes from arsenic binding were predicted to promote DNA-binding domain dissociation. Cys and structural destabilizing mutations were introduced. Recombinant technology was used to construct DNA for producing TetR variants redesigned for arsenic binding.Functional Evaluation of TetR Variants: To test arsenic responses, TetR variant plasmids and a DNA fragment containing the regulatory sequence tetO and gene sfgfp were co-introduced into Escherichia coli MG1655. Transformants were cultured 12 hours in LB medium with various arsenic concentrations. We measured the correlation between arsenic concentration and fluorescence intensity per 1.0 OD.Results: To develop an rsenic-sresponsive sensor, we first engineered a TetR variant (Variant 1) by introducing Cys substitutions into the DNA-binding domain of TetR. However, it showed no detectable fluorescence response upon arsenic exposure. Subsequently, additional destabilizing mutations were introduced into Variant 1 to create Variant 2. This variant showed a 2.8-fold increase in fluorescence at 10 ppm compared to the arsenic-free control. Furthermore, by increasing the number of arsenic-binding sites in Variant 3, the concentration required for maximal fluorescence shifted from 10 ppm to 5 ppm, indicqting a significant enhancement in sensitivity.Consideration: The lack of response in Variant 1 suggests that while arsenic binding may occur, it is insufficient to trigger the conformational change required for DNA dissociation. The successful induction observed in Variant 2 indicates that the introduction of destabilizing mutations effectively lowers the energetic barrier for this structural transition, thereby conferring arsenic responsiveness. Moreover, the improved sensitivity of Variant 3 demonstrates that increasing the number of binding sites is viable strategy for tuning the detection limit. These design, such as linking multiple TetR units, to develop biosensors capable of meeting more stringent environmental standards. Conclusion: In this study, we successfully converted TetR into an arsenic-responsive repressor by combing Cys substitutions with destabilizing mutations. Our results show that optimizing both the binding affinity and the allosteric transition is crucial for performance. We aim to achieve even higher sensitivity in future sensors.