Composite

Part:BBa_K4854027

Designed by: iGEM23_NYCU-Formosa   Group: iGEM23_NYCU-Formosa   (2023-09-03)

GFP+CsgA+Mfp5

This composite part contains lac promoter(BBa_R0010)[1], RBS(BBa_B0034)[2], GFP(BBa_E0040)[3], a flexible linker(BBa_K105012)[4], CsgA(BBa_K4854013)[5], GS linker(BBa_K4854012)[6] and Mfp5(BBa_K4854001)[7] gene. It is meant to express a fusion protein of the E. coli membrane protein CsgA, the mussel foot protein Mfp5, and the green fluorescent protein (GFP).
Mussel foot protein Mfp5 is an adhesive protein from the Mediterranean mussel (Mytilus galloprovincialis). Mussels can secrete special proteins to adhere to surfaces under turbulent environments.[8]
CsgA protein is the major component of biofilms of E. coli. It can self-assemble into a network of amyloid nanofibers outside the cell. Because of this characteristic, CsgA can be a platform to display recombinant protein on the surface of E. coli.[9]
We designed GFP as the functional protein linked at the N-terminal end of the CsgA+Mfp5 fusion protein.
We performed codon optimization to improve gene expression in E. coli.

circuit
Figure 1. The circuit of the GFP+CsgA+Mfp5 protein.


Cloning result

We successfully inserted GFP+CsgA+Mfp5 gene into pSB1A3 plasmid and amplified it in E. coli DH5α. After DNA sequencing, the plasmid was transformed into E. coli C41 for protein expression.

Gel electrophoresis result
Figure 2. Gel electrophoresis result.


Functional test

After expressed in E. coli C41 by 200 μM IPTG for 12 h at 37 ℃, we performed flushing test, viscosity test by rheometer, and modified ELISA to check whether GFP+CsgA+Mfp5 protein has adhesive properties.

1. Flushing test
With the flushing test, we can determine whether the functional adhesive recombinant protein was adhesive initially. We used our backbone pSB3K3+J04450, nature adhesive recombinant protein CsgA+Mfp5, and green fluorescence protein (GFP) as control. In contrast to the control, our functional adhesive recombinant protein had better adhesion. It could stick to slide marvelously.

functional test - Flushing test
Figure 3. Flushing test results.


2. Viscosity test
In Prof. Ming-Chia, Lee's lab, we used rheometer to further test the viscosity of functional adhesive recombinant protein GFP+CsgA+Mfp5. According to Figure 4, we can see that functional adhesive recombinant protein was 338.92% more adhesive than control J04450, 404.91% more adhesive than mussel recombinant protein, and 187.77% more adhesive than GFP. Functional adhesive recombinant protein is extremely adhesive.
functional test - Viscosity test
Figure 4. Viscosity test results. The y-axis is the percentage of the viscosity of the control and GFP+CsgA+Mfp5 divided by the viscosity of J04450 (control).


3. Modified ELISA
With the principle of ELISA antibody and antigen binding, we designed the modified ELISA to test whether our functional recombinant protein GFP+CsgA+Mfp5 had a great ability to capture antibodies. We replaced the antigen with the produced protein, used the viscosity of protein to capture the antigen and determined the strength of the binding antibody signal by OD630. We did a triple repeat and took the average value as the data. By Figure 5, we can tell that the functional adhesive recombinant protein had a better ability to capture antibodies than control J04450, CsgA+Mfp5 and GFP obviously.
functional test - Modified ELISA
Figure 5. The modified ELISA test triple repeat OD630 mean value of the control and GFP+CsgA+Mfp5. The y-axis is the percentage of the mean value of the proteins OD630 divided by the mean value of J04450 (control) OD630.

4. Fluorescence test
To verify that our functional adhesive recombinant protein could link together and allow the functional protein to function properly, we used a fluorescent microscope to observe the slides in the flushing test. According to Figure 6, GFP+CsgA+Mfp5 was adhesive while maintaining its functionality.
functional test - Fluorescence test
Figure 6. The fluorescent bacteria comparison between GFP and functional adhesive recombinant protein.


Reference
[1] https://parts.igem.org/Part:BBa_R0010
[2] https://parts.igem.org/Part:BBa_B0034
[3] https://parts.igem.org/Part:BBa_E0040
[4] https://parts.igem.org/Part:BBa_K105012
[5] https://parts.igem.org/Part:BBa_K4854013
[6] https://parts.igem.org/Part:BBa_K4854012
[7] https://parts.igem.org/Part:BBa_K4854001
[8] https://www.uniprot.org/uniprotkb/Q8WTE8/entry
[9] Fei Li, Luona Ye, Longyu Zhang, Xiaoyan Li, Xiaoxiao Liu, Jiarui Zhu, Huanhuan Li, Huimin Pang, Yunjun Yan, Li Xu, Min Yang, Jinyong Yan, Design of a genetically programmed barnacle-curli inspired living-cell bioadhesive, Materials Today Bio, Volume 14, 2022.


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
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 856


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