Part:BBa_K4854020

CsgA+Mrcp20k

This composite part contains lac promoter(BBa_R0010)^[1], RBS(BBa_B0034)^[2], CsgA(BBa_K4854013)^[3], GS linker(BBa_K4854012)^[4], and Mrcp20k(BBa_K4854006)^[5] gene. It is meant to express a fusion protein of the E. coli membrane protein CsgA and the barnacle cement protein Mrcp20k.
Barnacle cement protein Mrcp20k is an adhesive protein from the barnacle (Megabalanus rosa). At the cyprid stage before a barnacle becomes mature, the cyprid releases cement protein to anchor itself to substrates like rocks.^[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+Mrcp20k protein.

Cloning result

We successfully inserted CsgA+Mrcp20k 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+Mrcp20k 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 control. From Figure 3, we could make a preliminary decision that CsgA+Mrcp20k 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+Mrcp20k divided by the viscosity of J04450 (control), CsgA+Mrcp20k was 120.44% higher. We could tell that it was highly 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+Mrcp20k 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+Mrcp20k was 70.00% better than control (J04450). We could tell that the barnacle adhesive recombinant protein CsgA+Mrcp20k could 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_K4854006
[6] He, Li-Sheng et al. “Characterization of two 20kDa-cement protein (cp20k) homologues in Amphibalanus amphitrite." PloS one vol. 8,5 e64130. 22 May. 2013, doi:10.1371/journal.pone.0064130.
[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.

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
  COMPATIBLE WITH RFC[1000]