Composite

Part:BBa_K5151009

Designed by: KUO, YUN-HSIN   Group: iGEM24_NYCU-Formosa   (2024-08-23)

Lpp-OmpA-GS Linker(BELLO)-GFP-6x His Introduction

This composite part contains LacI promoter (BBa_R0010)[1], RBS (BBa_B0034)[2], Lpp-OmpA (BBa_K5151019)[3], GS linker (BBa_K5151018)[4] GFP (BBa_E0040)[5] 6x His (BBa_K3033006)[6] gene. It is meant to express a fusion protein of the E. coli membrane protein Lpp-OmpA, the Green Fluorescent Protein and the amino acid sequence tag 6x His.

Figure 1. The circuit of the Lpp-OmpA-GS Linker-GFP-6x His 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[7]. 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.
We plan to display specific proteins on the membrane using the Lpp-OmpA-GS linker. To validate the feasibility of this idea, we will use the Lpp-OmpA-GS linker to display a GFP-His on the membrane, allowing us to preliminarily confirm through ELISA that the protein expressed on the membrane is properly folded and functional[8].


Cloning Test

We cloned the insert, Lpp-OmpA-GS Linker-GFP-6x His, 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), 2-insert : Lpp-OmpA-GS Linker-GFP-6x His (1763 bp), 3-insert digest : Lpp-OmpA-GS Linker-GFP-6x His (1491 bp)

Function Test

After confirming the temperature effects from the literature[9], 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-6x 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.

Figure 3. ELISA result of Lpp-OmpA-GS Linker-GFP-6x His compared to bacteria with only J364007-pSB1C3.

Figure 3 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.

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_E0040
[6] Available at: https://parts.igem.org/Part:BBa_K3033006
[7] Available at: https://2024.igem.wiki/nycu-formosa/model
[8] Available at: https://2024.igem.wiki/nycu-formosa/design
[9] Highly efficient selection of phage antibodies mediated by display of antigen as Lpp-OmpA′ fusions on live bacteria. Available at: https://doi.org/10.1016/S0022-2836(00)94021-X

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
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1364


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