iGEM 2024 — Team nycu-formosa

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Week 1 (6/9-6/15) -

### Wet

Sequence optimization.

### Dry

Model design.

### HP

Discussed teaching content.

Week 2 (6/16-6/22) +

### Wet

Completed initial safety form.

Taught students experimental techniques.

### Dry

Established database.

### HP

Contacted teams in Taiwan.

Week 3 (6/23-6/29) +

### Wet

Sequence cloning.

Taught students experimental techniques.

### Dry

Encoding work.

### HP

Prepared briefing for Biotech Summer Camp.

Week 4 (6/30-7/6) +

### Wet

Cloning and sequencing.

### Dry

Cluster evaluation.

### HP

Taught students at Biotech Summer Camp.

Week 5 (7/7-7/13) +

### Wet

Cloning.

Lpp-OmpA-GFP protein expression testing.

### Dry

Migrated system to server.

### HP

Contacted WHU team.

Week 6 (7/14-7/20) +

### Wet

Learned ELISA.

Prepared high school student training.

### Dry

Ran NLP model to link disease and protein data.

### HP

Prepared briefing for NTHU.

Learned about blood glucose meter from JINHOO Technology.

Week 7 (7/21-7/27) +

### Wet

Conducted ELISA experiment, protein expression, and cloning.

### Dry

Optimized model and ensured data integrity.

### HP

Communicated with NTHU.

Taught high school students cloning experiments.

Week 8 (7/28-8/3) +

### Wet

Protein expression and ELISA experiment.

### Dry

Styled Wiki and began circuit diagram design.

### HP

Scheduled meetings with NTU and WHU teams.

Week 9 (8/4-8/10) +

### Wet

Cloning and ELISA experiment.

### Dry

Optimized circuit diagram.

Procured device materials

### HP

Communicated with NTU and WHU teams.

Week 10 (8/11-8/17) +

### Wet

Sequenced leukemia protein from model results.

### Dry

Constructed the device.

Procured device materials

### HP

Met with JINHOO Technology, Professor Yang Yu-Hsiung, and BioTik Technology.

Week 11 (8/18-8/24) +

### Wet

Learned electron current experiment.

Collected Wiki figures.

### Dry

Conducted small-scale probe testing.

Procured device materials

### HP

Communicated with CCU team.

Met with Professor Chao-Min Cheng; Created promotional video.

Week 12 (8/25-8/31) +

### Wet

Cloning (leukemia).

Optimized peptide sequence prediction.

### Dry

Conducted first chip test.

### HP

Met with APEXbio.

Uploaded promotional video.

Week 13 (9/1-9/7) +

### Wet

Cloning, protein expression, and ELISA experiment.

### Dry

Optimized device.

### HP

Conducted member photoshoot.

Compiled data and prepared copywriting.

Week 14 (9/8-9/14) +

### Wet

Protein expression, ELISA, and electron current experiment.

### Dry

Conducted second device test.

Week 15 (9/15-9/21) +

### Wet

Protein expression, ELISA, and electron current experiment.

### Dry

Document writing.

Week 16 (9/22-10/2) +

### Wet

Protein expression, ELISA, electron current experiment, and documentation.

### Dry

Wiki modification.

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![Button 1](https://static.igem.wiki/teams/5151/wiki/engineering/cycle1.png)

![Button 2](https://static.igem.wiki/teams/5151/wiki/engineering/cycle2.png)

![Button 3](https://static.igem.wiki/teams/5151/wiki/engineering/cycle3.png)

## Cycle 1 - Model establish

## Design

 The primary objective is to identify relationships between diseases and specific proteins and to develop a receptor capable of binding to these proteins. Initially, this involves collecting and analyzing existing associations between diseases and proteins to construct a preliminary disease-protein interaction network. Subsequently, the scope of diseases of interest and the types of proteins to be explored are defined, with particular emphasis on establishing the desired characteristics of the target receptor, such as binding affinity, specificity, and stability. At this stage, a comprehensive model architecture is devised, and appropriate feature engineering techniques are selected to establish a data-driven analytical framework.

---

## Build

 During the build phase, network biology approaches and natural language processing (NLP) are employed to perform text mining on articles from prestigious journals such as Nature and Cell to uncover potential relationships between diseases and proteins. A data processing pipeline is developed to extract disease-protein associations using NLP techniques. Concurrently, pretrained machine learning models are utilized to generate initial protein sequences, which serve as candidate receptors for binding to the target proteins.

---

## Test

 The discovered relationships between diseases and proteins are rigorously validated to ensure that the identified proteins are credible disease biomarkers. This verification process involves secondary literature reviews to confirm the association between the identified proteins and the diseases in question. In terms of receptor validation, molecular docking simulations are conducted to assess the binding affinity of the generated receptor sequences with the target proteins. Additionally, sequence alignment is performed to evaluate the similarity of the generated sequences with known functional receptors, ensuring structural and functional compatibility with the target proteins.

---

## Learn

 The outcomes from the testing phase are analyzed to identify and summarize the strengths and weaknesses of the model. Should inaccuracies be detected in the disease-protein associations, adjustments are made to the text mining algorithms or the literature dataset is expanded. If the generated receptor demonstrates suboptimal performance in binding assays, the receptor generation model is refined, or the sequence is optimized accordingly. Insights gained during this phase also guide the refinement of the disease database, ensuring that it exclusively comprises disease names, thus avoiding the inclusion of symptoms, to maintain data purity and analytical accuracy.

Next: Cycle 2 - Pretest

## Cycle 2 - Pretest

## Design

 From the 2015 NCTU Formosa Team, we gained insight into using Lpp-OmpA to express desired protein on the cell surface.[[1]](#reference)

 To verify this system, we designed a biobrick comprising Lpp-OmpA-GS Linker (we will refer to it as the “BELO system” from here on) fused to GFP-His. GFP was included to confirm surface expression via fluorescence, and the His tag was for later use with Anti-His antibodies in ELISA.

![The biobrick BELO-GFP-His BBa_K5151009 contains PLac promoter, RBS, Lpp-OmpA-GS Linker, GFP and 6X His tag](https://static.igem.wiki/teams/5151/wiki/engineering/figure-1-belo-gfp.png)

Figure 1. The biobrick BELO-GFP-His [BBa\_K5151009](https://parts.igem.org/Part%3ABBa_K5151009) contains PLac promoter, RBS, Lpp-OmpA-GS Linker, GFP and 6X His tag.

 The gene encoding Protein G was fused downstream of the BELO system to enable its surface expression on the bacterial outer membrane. Given that Protein G has an affinity for antibodies, its function was verified through ELISA by adding antibodies, and observing the changes in color and absorbance.

---

## Build

 We cloned the inserts, BELO-GFP, into the pSB1C3 vector and then transformed them into *E. coli* BL21 C41 cells. The reason for choosing pSB1C3 vector is that it is a well-known high-copy-number plasmid carrying a chloramphenicol resistance, allowing us to select for cells containing our inserts.[[2]](#reference) *E. coli* expressing BELO-Protein G was obtained from the team’s primary PI laboratory and used in ELISA experiments.

![The digest check results of the Lpp-OmpA-GS Linker (BELO) recombinant protein plasmid extracted from BL21 C41.](https://static.igem.wiki/teams/5151/wiki/engineering/figure-2-belo-gfp-parts-engineering-cloning.png)

Figure 2. The digest check results of the Lpp-OmpA-GS Linker (BELO) recombinant protein plasmid extracted from BL21 C41. M-Marker, 1-vector : J364007 - pSB1C3 (2029 bp), 2-insert : Lpp-OmpA-GS Linker-GFP (1763 bp), 3-insert digest : Lpp-OmpA-GS Linker-GFP (1491 bp)

---

## Test

#### ELISA

 To test whether our devices can capture antibodies or related substances, we used two types of enzyme-linked immunosorbent assay (ELISA) to confirm the system. The first was a direct ELISA using protein G, where different concentrations of Goat Anti-Rabbit conjugated with HRP were added and allowed to react with 3,3',5,5'-tetramethylbenzidine (TMB). The second was sandwich ELISA with a slight modification. Since the His-tag was expected to be expressed on the cell surface, we coated the plate with *E. coli* instead of capturing antibodies. The secondary antibody used was Anti-His conjugated with HRP, and TMB was used again as the chromogenic substrate.

![](https://static.igem.wiki/teams/5151/wiki/engineering/new-figure-3-lpp-ompa-gs-linker-protein-g-goat-anti-rabbit.png)

Figure 3. ELISA result of Lpp-OmpA-GS-protein G with different concentrations of Goat Anti-rabbit.

 Figure 3 shows that the OD630 values increased proportionally with the concentration of Goat Anti-Rabbit.

 After confirming the temperature effects from the literature[[3]](#reference), we conducted the test at 16°C for 18 hours. We evaluated the expression of BELO-GFP with 6X His tag on the outer membrane. To achieve this, we conducted a comparative analysis using sandwich ELISA between *E. coli* with only the pSB1C3 alone (containing GFP) and *E. coli* containing BELO-GFP-His.

 We hypothesized that successful expression and functional integration of BELO-GFP with 6X His on the outer membrane would result in ELISA outcomes that positively correlate with the Anti-His antibody concentration, exhibiting a more significant signal compared to the control bacteria containing only the vector.

![ELISA result of BELO-GFP-His compared to bacteria with only J364007-pSB1C3](https://static.igem.wiki/teams/5151/wiki/engineering/new-figure-4-lpp-ompa-gs-linker-gfp-his-anti-his.png)

Figure 4. ELISA result of BELO-GFP-His compared to bacteria with only J364007-pSB1C3.

 Figure 4 illustrates the significant differences in ELISA results at OD630 between *E. coli* strains with and without the BELO system. At each concentration of Anti-His antibody, *E. coli* expressing the BELO system exhibited higher OD630 values compared to the control strain. This indicates that the BELO system effectively facilitates the expression of the target protein on the outer membrane, enabling specific molecular binding.

---

## Learn

 In this round of experiments, we confirmed that the BELO system successfully anchored proteins, such as protein G, to the outer membrane, with only minor limitations regarding protein solubility.[[4]](#reference)

Next: Cycle 3 - Predicted Protein

## Cycle 3 - Predicted Protein

## Design

 After verifying that the BELO system can display proteins on the cell surface, we moved on to the adhesion G protein-coupled receptor CD97[[5][6]](#reference), which our model selected for its potential in rapid detection. We designed a biobrick to express its ligand, complement decay-accelerating factor CD55[[7]](#reference), with the aim of applying it to the detection of Acute Myeloid Leukemia. This construct also serves as our positive control to ensure the reliability of our detection system in this round of experiments.

![The biobrick BELO-CD55 BBa_K5151010 contains PLac promoter, RBS, Lpp-OmpA-GS Linker and CD55](https://static.igem.wiki/teams/5151/wiki/engineering/figure-5-belo-cd55.png)

Figure 5. The biobrick BELO-CD55 [BBa\_K5151010](https://parts.igem.org/Part%3ABBa_K5151010) contains PLac promoter, RBS, Lpp-OmpA-GS Linker and CD55.

Additionally, we designed a biobrick for CD97-His to be used in the modified sandwich ELISA. In this setup, CD97-His will bind to CD55 expressed on *E. coli* via its CD97 domain, while Anti-His conjugated with HRP will bind to the His-tag for detection.

![The concept of the modified sandwich ELISA](https://static.igem.wiki/teams/5151/wiki/engineering/figure-6-elisa-concept.png)

Figure 6. The concept of the modified sandwich ELISA.

![The biobrick CD97-His BBa_K5151013 contains PLac promoter, RBS, CD 97 and His-tag](https://static.igem.wiki/teams/5151/wiki/engineering/figure-7-cd97-his.png)

Figure 7. The biobrick CD97-His [BBa\_K5151013](https://parts.igem.org/Part%3ABBa_K5151013) contains PLac promoter, RBS, CD97 and His-tag.

 However, not all biomarkers have receptors that can bind to them. To address this, we used PeptiMap to predict short peptide sequences that could potentially bind to CD97, followed by local alignment with the known CD55 receptor. We then utilized iGEMDOCK to simulate the binding positions and modes between CD97 and the peptides. Through this process, we identified two peptide sequences, 2BOU-2 and 2BOU-3. These were designed to be fused with Lpp-OmpA using a GS linker, resulting in BELO-2BOU-2 and BELO-2BOU-3. The constructs were cloned into the pSB1C3 plasmid and expressed on the outer membrane of *E. coli*. An ELISA assay was then performed to validate the binding functionality of these constructs with CD97-His.

![The biobrick BELO-2BOU-2 BBa_K5151006 contains PLac promoter, RBS, Lpp-OmpA-GS Linker and short peptide, 2BOU-2](https://static.igem.wiki/teams/5151/wiki/engineering/figure-8-belo-2bou-2.png)

Figure 8. The biobrick BELO-2BOU-2 [BBa\_K5151006](https://parts.igem.org/Part%3ABBa_K5151006) contains PLac promoter, RBS, Lpp-OmpA-GS Linker and short peptide, 2BOU-2.

![The biobrick BELO-2BOU-3 BBa_K5151015 contains PLac promoter, RBS, Lpp-OmpA-GS Linker and short peptide, 2BOU-3](https://static.igem.wiki/teams/5151/wiki/engineering/figure-9-belo-2bou-3.png)

Figure 9. The biobrick BELO-2BOU-3 [BBa\_K5151015](https://parts.igem.org/Part%3ABBa_K5151015) contains PLac promoter, RBS, Lpp-OmpA-GS Linker and short peptide, 2BOU-3.

---

## Build

 We cloned four inserts, BELO-CD55, CD97-His, BELO-2BOU-2, and BELO-2BOU-3 into the pSB1C3 plasmid and transformed them into *E. coli* BL21 C41.

![The digest check results of the Lpp-OmpA-GS Linker (BELO) recombinant protein plasmid extracted from BL21 C41.](https://static.igem.wiki/teams/5151/wiki/engineering/figure-10-belo-cd55-cd97-his-engineering-cloning.png)

Figure 10. The digest check results of the Lpp-OmpA-GS Linker (BELO) recombinant protein plasmid extracted from BL21 C41. M-Marker, 4-insert : Lpp-OmpA-GS Linker-CD55 (2067 bp), 5-insert digest : Lpp-OmpA-GS Linker- CD55 (1895 bp), 6-insert : CD97-His (1326 bp), 7-insert digest : CD97-His (1054 bp)

![The digest check results of the Lpp-OmpA-GS Linker (BELO) recombinant protein plasmid extracted from BL21 C41.](https://static.igem.wiki/teams/5151/wiki/engineering/figure-11-belo-2bou-engineering-cloning.png)

Figure 11. The digest check results of the Lpp-OmpA-GS Linker (BELO) recombinant protein plasmid extracted from BL21 C41. M-Marker, 1-vector : J364007 - pSB1C3 (2029 bp), 2-insert : Lpp-OmpA-GS Linker-2BOU-2 (1062 bp), 3-insert digest : Lpp-OmpA-GS Linker-2BOU-2 (791 bp), 4-insert : Lpp-OmpA-GS Linker-2BOU-3 (1044 bp), 5-insert digest : Lpp-OmpA-GS Linker-2BOU-3 (772 bp)

---

## Test

 The goal of our ELISA experiment is to assess the receptor's ability to bind to the target protein. After coating the target receptor and performing blocking, different concentrations of CD97-His are added. This is followed by the addition of an enzyme-labeled secondary antibody, Anti-His-HRP, and the enzyme substrate TMB for color development. Finally, OD630 is measured to verify the binding capacity of the target receptor to CD97-His.

 We performed a sandwich ELISA to test whether CD55, 2BOU-2 and 2BOU-3 were successfully anchored to the cell membrane at 16°C for 18 hr.

![The ELISA result of two prediction peptides with negative control and positive control binding with CD97-His.](https://static.igem.wiki/teams/5151/wiki/engineering/new-figure-12-different-receptors-with-cd97-his.png)

Figure 12. The ELISA result of two prediction peptides with negative control and positive control binding with CD97-His. From left to right: J364007-pSB1C3 (negative control), Lpp-OmpA-GS Linker-CD55 (positive control), Lpp-OmpA-GS Linker-2BOU-2 and Lpp-OmpA-GS Linker-2BOU-3.

 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.

 Comparing the ELISA results of 2BOU-2 and 2BOU-3 suggests that 2BOU-3 has a better affinity for binding with CD97-His. Consequently, we chose BELO-2BOU-3 to test the binding capacity with different CD97-His dilution ratios.

![ELISA result of BELO-2BOU-3 compared to bacteria with only J364007-pSB1C3](https://static.igem.wiki/teams/5151/wiki/engineering/new-figure-13-lpp-ompa-gs-linker-2bou-3-cd97-his.png)

Figure 13. ELISA result of BELO-2BOU-3 compared to bacteria with only J364007-pSB1C3.

 Figure 13 illustrates a positive correlation between increasing concentrations of anti-His antibodies conjugated with HRP and OD630. This indicates that the BELO system can effectively express the predicted peptide 2BOU-3 with functional capabilities and detect it concentration-dependent, thereby validating its potential as a biosensing tool for this biomarker.

---

## Learn

 In this cycle, we verified that our devices effectively capture CD97-His, strengthening our confidence in their potential as a rapid detection method for Acute Myeloid Leukemia when applied on the chip. This may provide an additional assessment tool to support diagnosis.

## Reference

1. NCTU Formosa. (n.d.). *Team NCTU Formosa.* iGEM. [https://2015.igem.org/Team:NCTU\_Formosa](https://2015.igem.org/Team%3ANCTU_Formosa)
2. iGEM Registry. (n.d.). *Part: pSB1C3.* iGEM. [https://parts.igem.org/Part:pSB1C3](https://parts.igem.org/Part%3ApSB1C3)
3. Highly efficient selection of phage antibodies mediated by display of antigen as Lpp-OmpA′ fusions on live bacteria [https://doi.org/10.1016/S0022-2836(00)94021-X](https://doi.org/10.1016/S0022-2836%2800%2994021-X)
4. Production and fluorescence-activated cell sorting of Escherichiacoli expressing a functional antibody fragment on theexternal surface <https://doi.org/10.1073/pnas.90.22.10444>
5. Stacey M, Yona S (2011). *Adhesion-GPCRs: Structure to Function (Advances in Experimental Medicine and Biology).* Berlin: Springer. ISBN 978-1-4419-7912-4.
6. Langenhan T, Aust G, Hamann J (May 2013). "Sticky signaling--adhesion class G protein-coupled receptors take the stage". Science Signaling. 6 (276): re3. [doi:10.1126/scisignal.2003825.](https://doi.org/10.1126/scisignal.2003825) PMID 23695165. S2CID 6958640.
7. Ruiz-argüelles, A., & Llorente, L. (2007). The Role of Complement Regulatory Proteins (CD55 and CD59) in the Pathogenesis of Autoimmune Hemocytopenias. *Autoimmunity Reviews*, 6(3), 155–161. <https://www.sciencedirect.com/science/article/abs/pii/S1568997206001637?via%3Dihub>

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# Click button on the top to see different protocol.

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Cloning
Protein expression
ELISA
Electric current experiment
Hardware CV testing

## Protocol for doing cloning

## Protocol for protein expression

## Protocol for ELISA

## Protocol for electric current experiment

## This is the protocol to use our hardware

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## 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.
![BELO-CD55](https://static.igem.wiki/teams/5151/wiki/results/belo-cd55.png)
![BELO-2BOU-2](https://static.igem.wiki/teams/5151/wiki/results/belo-2bou-2.png)
![BELO-2BOU-2](https://static.igem.wiki/teams/5151/wiki/results/belo-2bou-2.png)

Figure 1. Biobricks design of BELO system.

We also express our biomarker by ourselves.
![CD97-His](https://static.igem.wiki/teams/5151/wiki/results/cd97-his.png)

Figure 2. Biobrick design of biomarkers.

## Cloning results

We successfully inserted the biobricks into the vector, J364007-pSB1C3, amplified our plasmids in *E. coli* BL21 C41, and expressed them.
![clonining results of GFP/CD55](https://static.igem.wiki/teams/5151/wiki/results/figure3.png)

Figure 3. The digest check results of the BELO (Lpp-OmpA-GS Linker) recombinant protein plasmid extracted from BL21 C41. M-Marker, 1-vector : J364007 - pSB1C3 (2029 bp), 2-insert : Lpp-OmpA-GS Linker-GFP (1763 bp), 3-insert digest : Lpp-OmpA-GS Linker-GFP (1491 bp), 4-insert : Lpp-OmpA-GS Linker-CD55 (2067 bp), 5-insert digest : Lpp-OmpA-GS Linker-CD55 (1895 bp), 6-insert : CD97-His (1326 bp), 7-insert digest : CD97-His (1054 bp).

![clonining results of GFP/CD55](https://static.igem.wiki/teams/5151/wiki/results/figure4.png)

Figure 4. The digest check results of the BELO (Lpp-OmpA-GS Linker) recombinant protein plasmid extracted from BL21 C41. M-Marker, 1-vector : J364007 - pSB1C3 (2029 bp), 2-insert : Lpp-OmpA-GS Linker-2BOU-2 (1062 bp), 3-insert digest : Lpp-OmpA-GS Linker-2BOU-2 (791 bp), 4-insert : Lpp-OmpA-GS Linker-2BOU-3 (1044 bp), 5-insert digest :Lpp-OmpA-GS Linker-2BOU-3 (772 bp).

---

## 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.

![ELISA concept](https://static.igem.wiki/teams/5151/wiki/results/figure5-elisa-concept.png)

Figure 5. The concept of the modified sandwich ELISA.

![ELISA result from protein G bind with Goat Anti-Rabbit antibody](https://static.igem.wiki/teams/5151/wiki/results/figure6-lpp-ompa-gs-linker-protein-g-goat-anti-rabbit.png)

Figure 6. ELISA results from protein G bind with Goat Anti-Rabbit antibody.

 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.

![ELISA result of BELO-GFP-His compared to bacteria with only J364007-pSB1C3](https://static.igem.wiki/teams/5151/wiki/results/figure7-lpp-ompa-gs-linker-gfp-his-anti-his.png)

Figure 7. ELISA result of BELO-GFP-His compared to bacteria with only J364007-pSB1C3.

 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.

![ELISA results of BELO-CD55, BELO-2BOU-2, BELO-2BOU-3 compared to bacteria with only J364007-pSB1C3 bind with CD97-His and anti-His-HRP](https://static.igem.wiki/teams/5151/wiki/results/figure8-different-receptors-with-cd97-his.png)

Figure 8. ELISA results of BELO-CD55, BELO-2BOU-2, BELO-2BOU-3 compared to bacteria with only J364007-pSB1C3 bind with CD97-His and anti-His-HRP.

 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.

![ELISA result of BELO-2BOU-3 compared to bacteria with only J364007-pSB1C3 binding with CD97-His and anti-His-HRP](https://static.igem.wiki/teams/5151/wiki/results/figure9-lpp-ompa-gs-linker-2bou-3-cd97-his.png)

Figure 9. ELISA result of BELO-2BOU-3 compared to bacteria with only J364007-pSB1C3 binding with CD97-His and anti-His-HRP.

 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

![The current change results from the binding of the BELO (Lpp-OmpA-GS Linker) recombinant protein and the J364007-pSB1C3 with CD97-His.](https://static.igem.wiki/teams/5151/wiki/results/new-figure10.png)

Figure 10. The current change results from the binding of the BELO (Lpp-OmpA-GS Linker) recombinant protein and the J364007-pSB1C3 with CD97-His. (Negative control : J364007-pSB1C3, positive control : BELO-CD55)

 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.

![The current change results from the binding of Lpp-OmpA-GS Linker-2BOU-3 with CD97-His](https://static.igem.wiki/teams/5151/wiki/results/new-figure11.png)

Figure 11. The current change results from the binding of Lpp-OmpA-GS Linker-2BOU-3 with CD97-His.

 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.

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## 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.

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