Part:BBa_K5151015

Lpp-OmpA-GS Linker(BELLO)-2BOU-3 Introduction

This composite part contains LacI promoter (BBa_R0010)^[1], RBS (BBa_B0034)^[2], Lpp-OmpA (BBa_K5151019)^[3], GS linker (BBa_K5151018)^[4] 2BOU-3 (BBa_K5151004)^[5] gene. It is meant to express a fusion protein of the E. coli membrane protein Lpp-OmpA and the peptide 2BOU-3.

Figure 1. The circuit of the Lpp-OmpA-GS Linker-2BOU-3 protein.

Project Introduction

Early detection of diseases is crucial. To achieve this, we can target disease biomarkers for testing to assist in early diagnosis. Currently, clinical tests for disease biomarkers, such as ELISA, RIA, and mass spectrometry, though widely used, tend to require longer detection times. Many have developed biosensors and rapid diagnostic tools (RDT). However, challenges still exist in detecting disease biomarkers, mainly due to the complexity of samples and the insufficient sensitivity and specificity of detection technologies.
To address these issues, our team has proposed a new strategy this year, 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 technologies and provide a more efficient and cost-effective pathway for early diagnosis and timely treatment of diseases. This not only helps to shorten diagnostic times but also improves diagnostic accuracy and patient treatment 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.
In our project, we hope to determine the expression level of CD97 through electrochemical detection, which requires us to find the receptor for CD97. In addition to the CD55 protein found in literature, we also introduced Generative AI to design short peptides that can bind to potential biomarkers, specifically 2BOU-3. We will use these peptides in the detection system to enhance its functionality. Subsequently, we attached 2BOU-3 to the Lpp-OmpA-GS Linker sequence and transformed plasmids containing the Lpp-OmpA-GS Linker-2BOU-3 sequence into E. coli for expression, allowing 2BOU-3 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^[7].

Analysis results

The following peptide structures have been generated using PEP-FOLD 4.0:

Figure 2. The structure of 2BOU-3.

CD97 is shown in green, the peptides in blue and the interacting residues of CD97 in dark green.

Figure 3. The docking result of 2BOU-3 and CD97.

According to the iGEMDOCK results, 2BOU-3 interacts with residues such as Cys-47, Ala-48, Ser-54, Cys-55, Ser-59, Asp-60, Cys-61, Trp-62.
Melania Capasso, Lindy G. Durrant, and Martin Stacey have discovered that CD55 would bind with CD97. Thus, we took CD55 to compare with our result.
The results of the local alignment are shown in the following figures:

Figure 4. The local alignment result of 2BOU-3 and CD55 with the sequence of 2BOU-3.

2BOU-3 did not meet the minimum peptide length requirement for alignment, so there is no result for 2BOU-3.

Cloning Test

We cloned the insert, Lpp-OmpA-GS Linker-2BOU-3, into the pSB1C3 plasmid and transformed them into Escherichia coli BL21 C41.

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-2BOU-3 (1044 bp), 5-insert digest : Lpp-OmpA-GS Linker-2BOU-3 (772 bp).

Function Test

ELISA Results

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-2BOU-3 compared to bacteria with only J364007-pSB1C3.

Figure 4. The ELISA result of Lpp-OmpA-GS Linker-2BOU-3 compared to bacteria with J364007-pSB1C3 at different CD97-6x His dilution ratios.

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 5. The current change results from the binding of the BELO (Lpp-OmpA-GS Linker) recombinant protein and the J364007-pSB1C3 with CD97-6x His.

Figure 6. The current change results from the binding of the BELO (Lpp-OmpA-GS Linker) recombinant protein with different CD97-6x His dilution ratios.

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_K5151004
[6] Available at: https://2024.igem.wiki/nycu-formosa/model
[7] 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 617
  
• 1000
  COMPATIBLE WITH RFC[1000]