Contribution

Figure 1: Backbone plasmids used for composite part construction. Left: pGL4.35 (with 9×GAL4 UAS and luciferase reporter); Right: pcDNA3.1 (with CMV promoter for high expression).

Figure 2: The basic parts we used include 13 types such as TEV components, Lac-sensor components, Linker, VP-64TAD, TEV-cs, Flag, and other regulatory elements. These basic parts were utilized to construct our composite parts.

Figure 3: This figure illustrates the design of our composite parts using the uploaded basic parts, which include the lactate-sensing components (LS1.0 and Stev-lac-0.1 to Stev-lac-0.6). These specific composite parts serve as the lactate-sensing module for constructing our system.

Figure 4: This figure illustrates the design of our composite parts using the uploaded basic parts, including partial lactate-sensing components (Stev-lac-0.7 to Stev-lac-0.8), the lactate-degrading component (sLOx), and the regulatory component (GV2ER). Together with the lactate-sensing composite parts shown in the previous figure, these components were used to construct our entire system.

Figure 5: GV-2ER: The "G" stands for the yeast Gal4 DNA-binding domain. In transcriptional regulation, this domain can specifically recognize and bind to DNA sequences containing Gal4 response elements. The "V" represents the herpes simplex virus VP64 transactivation domain, which possesses a potent function of activating transcription.

Figure 6: This figure presents the map of the novel plasmid designed for verifying sLOx function. On the basis of the original plasmid used in sensor screening (which contained the Luciferase reporter gene), the Luciferase gene was replaced with the optimized sLOx gene. The plasmid retains key elements required for gene expression and cell screening, enabling the expression of sLOx to be regulated by the upstream lactate sensor system and facilitating subsequent verification of sLOx secretion and lactate-degrading activity.

Figure 7: This figure presents the background screening results of the LS3.1-LS3.8 sensors. The ordinate (RLU) reflects the TEV enzyme activity of each sensor without lactate stimulation; the results help screen out sensors with low background activity (e.g., LS3.5, LS3.6, LS3.8).

Figure 8: This figure shows the sensitivity and stability verification results of the screened low-background sensors (LS3.5, LS3.6, LS3.8) under different lactate concentrations (0mM, 1mM, 5mM). The ordinate (RLU) indicates TEV enzyme activity; the results confirm that LS3.5 has the highest sensitivity and stability, as it shows a more obvious response to lactate concentration changes while maintaining low background.

Figure 9: This figure displays the results of the Western blot assay used to verify sLOx protein secretion. The blot includes two sample types: culture medium (extracellular fluid) and cell lysate (intracellular fluid), with both NC (negative control) and LS3.5 (sensor-sLOx system) groups tested. The presence of the sLOx protein band in the culture medium of the LS3.5 group (while no obvious band is detected in the NC group's culture medium) confirms that the engineered sLOx protein was successfully secreted from 293T cells into the extracellular space.

Figure 10: This figure shows the results of the extracellular lactate degradation assay. The ordinate represents the relative concentration of extracellular lactate, and the abscissa shows the NC group and the LS3.5 group (sensor-sLOx system group). The results clearly indicate that compared with the NC group, the extracellular lactate concentration in the LS3.5 group is significantly lower, directly demonstrating that the secreted sLOx protein has a stable and efficient lactate-degrading function, which can reduce the local extracellular lactate concentration as expected.