Part:BBa_K4854014

CsgA+Mfp3

This composite part contains lac promoter(BBa_R0010)^[1], RBS(BBa_B0034)^[2], CsgA(BBa_K4854013)^[3], GS linker(BBa_K4854012)^[4] and Mfp3(BBa_K4854000)^[5] gene. It is meant to express a fusion protein of the E. coli membrane protein CsgA and the mussel foot protein Mfp3.
Mussel foot protein Mfp3 is an adhesive protein from the Mediterranean mussel (Mytilus galloprovincialis). Mussels can secrete special proteins to adhere to surfaces under turbulent environments.^[6]
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.^[7]
We performed codon optimization to improve gene expression in E. coli.

Figure 1. The circuit of the CsgA+Mfp3 protein.

Cloning result

We successfully inserted CsgA+Mfp3 gene into pSB1A3 plasmid and amplified it in E. coli DH5α. After DNA sequencing, we replaced the vector pSB1A3 with pSB3K3 for better protein expression. Then the plasmid was transformed into E. coli C41 for protein expression.

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 CsgA+Mfp3 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 as the control. From Figure 3, we could make a preliminary decision that CsgA+Mfp3 did not had obvious adhesion to stick on the slides.

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.Taking the percentage of the viscosity of CsgA+Mfp3 divided by the viscosity of J04450 (control), CsgA+Mfp3 was 2.54% higher. We could tell that it was not really adhesive.

Figure 4. The viscosity test results of natural adhesive recombinant proteins. The y-axis is the percentage of the viscosity of the natural adhesive recombinant proteins 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 CsgA+Mfp3 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. Based on Figure 5, the ability of capturing the antibodies of CsgA+Mfp3 was 13.47% lower than control (J04450). We could tell that our mussel adhesive recombinant protein CsgA+Mfp3 could not capture antibodies effectively.

Figure 5. The modified ELISA test triple repeat OD₆₃₀ mean value of natural adhesive recombinant proteins. The y-axis is the percentage of the mean value of the natural adhesive recombinant protein OD₆₃₀ divided by the mean value of J04450 (control) OD₆₃₀.

Reference
[1] https://parts.igem.org/Part:BBa_R0010
[2] https://parts.igem.org/Part:BBa_B0034
[3] https://parts.igem.org/Part:BBa_K4854013
[4] https://parts.igem.org/Part:BBa_K4854012
[5] https://parts.igem.org/Part:BBa_K4854000
[6] Zhong, C., Gurry, T., Cheng, A. et al. Strong underwater adhesives made by self-assembling multi-protein nanofibres. Nature Nanotech 9, 858–866 (2014).
[7] 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.