Overview
To establish our BELO system (Lpp-OmpA-GS Linker), we constructed five proteins after BELO to express as outer membrane proteins, allowing us to test the system's functionality. We conducted modified ELISA and current-based experiments to assess the performance of these proteins. The results demonstrated that the Lpp-OmpA-GS Linker successfully facilitated the expression of proteins on the outer membrane, while maintaining their functionality outside the membrane. Additionally, we confirmed that these proteins can be effectively detected using electrochemical biosensors.
Biobrick design
Our sequences of BELO were organized by Lpp-OmpA-GS Linker recombinant with receptor proteins.
We also express our biomarker by ourselves.
Cloning results
We successfully inserted the biobricks into the vector, J364007-pSB1C3, amplified our plasmids in E. coli BL21 C41, and expressed them.
ELISA result
With the principle of antibody-antigen binding in ELISA, we designed a modified sandwich ELISA to test whether the proteins expressed by the BELO system had a great ability to capture target proteins.
We replaced the antigen with the BELO (Lpp-OmpA-GS Linker)-receptor, used the membrane-expressed proteins to capture biomarkers, and determined the strength of the binding protein signal at OD630. We did a triple repeat, and took the average value as the data.
According to Figure 6, the ELISA results demonstrated a positive correlation between increasing concentrations of Goat Anti-rabbit and OD630. As the concentration of Goat Anti-rabbit increases, more molecules are able to bind, resulting in a higher OD630 value. This indicates that the BELO system can effectively express the protein G with functional capabilities and detect it concentration-dependent.
According to Figure 7, the ELISA results demonstrated a positive correlation between increasing concentrations of anti-His antibody and OD630. As the concentration of anti-His antibodies increases, more molecules are able to bind to BELO-GFP-His, resulting in a higher OD630 value. This indicates that the BELO system can effectively express the BELO-GFP-His with functional capabilities and detect it concentration-dependent.
According to Figure 8, the bar chart illustrates that the positive control exhibits the highest OD630 value, while the negative control shows the lowest OD630 value. This indicates that both 2BOU-2 and 2BOU-3 effectively bind to CD97-His. Additionally, the higher OD630 value observed for BELO-2BOU-3 compared to BELO-2BOU-2 suggests that 2BOU-3 has a superior affinity for CD97-His.
According to Figure 9, the ELISA results demonstrated a positive correlation between increasing concentrations of anti-His antibody and OD630. As the concentration of CD97-His increases, more molecules are able to bind to 2BOU-3, resulting in a higher OD630 value.
This indicates that the BELO system can effectively express the predicted peptide 2BOU-3 with functional capabilities and detect it concentration-dependent.
Current experiment results
With the principle of antibody-antigen binding in ELISA, we designed a current experiment based on a modified sandwich ELISA to test whether the proteins expressed by BELO had a great ability to capture target proteins.
We added TMB as the final reaction and then used an electrochemical biosensor to sense the amount of electrons produced by the redox reaction.
Overview of the current change of four sequences
According to Figure 10, we can see that the current change of BELO recombinant protein was more significant than the control (J364007-pSB1C3) without BELO system. By measuring the current change, we can indirect proof that the BELO system had the ability to capture biomarkers.
BELO-2BOU-3 with different CD97-His dilution ratio
Compare between BELO-2BOU-2 and BELO-2BOU-3, we can observe that the ability of capturing the CD97-His protein of BELO-2BOU-3 is better than BELO-2BOU-2, so the next experiment is test the current of BELO-2BOU-3 with different CD97-His dilution ratio.
According to Figure 11, the current change of BELO-2BOU-3 decreases as the CD97-His concentration decreases, indicating that the electrochemical response of our system is dependent on the concentration of CD97-His. This suggests a direct correlation between the binding of CD97-His to the BELO-2BOU-3 system and the observed current change. As the concentration of CD97-His decreases, fewer molecules are available to bind, resulting in a smaller current change. This demonstrates that our system is sensitive to varying concentrations of CD97-His and can effectively detect it in a concentration-dependent manner, validating its use as a biosensing tool for this biomarker.
Conclusion
The results of the two functional tests provided comprehensive insights. First, the BELO system successfully expressed the proteins as outer membrane components and demonstrated the ability to capture biomarkers. Second, all of our predicted receptor proteins were functional. Additionally, our electrochemical detection method clearly distinguished between bound and unbound states. These findings confirmed that our CHELO concept can be practically implemented, allowing for the successful expression of both peptides and proteins on the outer membrane to bind with the target protein.