Part:BBa_K5151010

Lpp-OmpA-GS Linker(BELLO)-CD55 Introduction

This composite part contains LacI promoter (BBa_R0010)^[1], RBS (BBa_B0034)^[2], Lpp-OmpA (BBa_K5151019)^[3], GS linker (BBa_K5151018)^[4] CD55 (BBa_K4854000)^[5] gene. It is meant to express a fusion protein of the E. coli membrane protein Lpp-OmpA and the glycoprotein CD55.

Figure 1. The circuit of the Lpp-OmpA-GS Linker-CD55 protein.

Project Introduction

Early detection of diseases is crucial. To achieve this, we can focus on detecting disease biomarkers to aid in early diagnosis. Currently, clinical tests for disease biomarkers, such as ELISA, RIA, and mass spectrometry, are widely used but often require lengthy detection times. Many researchers have developed biosensors and rapid diagnostic tools (RDTs). However, there are still numerous challenges in detecting disease biomarkers, primarily due to the complexity of samples and the limitations in sensitivity and specificity of the detection technologies.
To address these issues, our team has proposed a new strategy this year by utilizing a pre-trained Natural Language Processing (NLP) model to identify potential biomarkers for diseases^[6]. Through this approach, we aim to overcome the current bottlenecks in detection technology, providing a more efficient and cost-effective method for early diagnosis and timely treatment of diseases. This not only helps shorten diagnosis time but also improves diagnostic accuracy and patient outcomes.
Ultimately, our model identified the top ten highly relevant diseases, and we selected leukemia as the target for rapid detection to demonstrate the feasibility of our strategy. Based on the model’s results, we chose CD97 from all proteins associated with leukemia as the biomarker. Subsequently, we aim to use an electrochemical detector to measure the expression level of CD97 to determine whether a patient has the disease.
To determine the expression level of CD97 through electrochemical detection, we need to find the receptor for CD97. Therefore, we identified a protein that can bind to CD97 from the literature, which is CD55^[7]. CD55, also known as decay-accelerating factor (DAF), is a membrane protein encoded by the CD55 gene that is anchored by a GPI tail^[8]. Research shows that the four short consensus repeat (SCR) domains at the N-terminus of CD55 can bind to three to five epidermal growth factor (EGF) domains at the N-terminus of CD97^[9].
In our project, we will attach CD55 to the Lpp-OmpA-GS Linker sequence and transform plasmids containing the Lpp-OmpA-GS Linker-CD55 sequence into E. coli for expression, allowing CD55 to be displayed on the surface of E. coli. When it binds with CD97 present in the sample, we can obtain an electrochemical signal from the binding. Finally, we will analyze the binding electrochemical signals to determine whether the patient has the disease^[10].

Cloning Test

We cloned the insert, Lpp-OmpA-GS Linker-CD55, into the pSB1C3 plasmid and transformed them into Escherichia coli BL21 C41.

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), 4-insert : Lpp-OmpA-GS Linker-CD55 (2067 bp), 5-insert digest : Lpp-OmpA-GS Linker- CD55 (1895 bp).

Function Test

ELISA Test

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.

Figure 3. ELISA result of Lpp-OmpA-GS Linker-CD55 compared to bacteria with only J364007-pSB1C3.

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.

Figure 4. The current change results from the binding of the BELO (Lpp-OmpA-GS Linker) recombinant protein and the J364007-pSB1C3 with CD97-His.

Reference

[1] Available at: https://parts.igem.org/Part:BBa_R0010
[2] Available at: https://parts.igem.org/Part:BBa_B0034
[3] Available at: https://parts.igem.org/Part:BBa_K5151019
[4] Available at: https://parts.igem.org/Part:BBa_K5151018
[5] Available at: https://parts.igem.org/Part:BBa_K5151000
[6] Available at: https://2024.igem.wiki/nycu-formosa/model
[7] Hamann, J., Vogel, B., van schijndel, G. m, & Van lier, R. a. (1996). The Seven-Span Transmembrane Receptor CD97 Has a Cellular Ligand (CD55, DAF). J Exp Med, 184(3), 1185–1189. Available at: https://rupress.org/jem/article-abstract/184/3/1185/50946/The-seven-span-transmembrane-receptor-CD97-has-a?redirectedFrom=fulltext
[8] 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. Available at: https://www.sciencedirect.com/science/article/abs/pii/S1568997206001637?via%3Dihub
[9] Niu, M., Xu, shengzhao, Yang, J., Yao, D., Li, N., Yang, J., Zhong, G., & Song, G. (2021). Structural Basis for CD97 Recognition of the Decay-Accelerating Factor CD55 Suggests Mechanosensitive Activation of Adhesion GPCRs. Journal Biological Chemistry, 296. Available at: https://www.jbc.org/article/S0021-9258(21)00569-X/fulltext
[10] Available at: https://2024.igem.wiki/nycu-formosa/index.html

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 616
  
• 1000
  COMPATIBLE WITH RFC[1000]