Part:BBa_K4854021
CsgA+Aacp19k
This composite part contains lac promoter(BBa_R0010)[1],
RBS(BBa_B0034)[2],
CsgA(BBa_K4854013)[3],
GS linker(BBa_K4854012)[4]
and Aacp19k(BBa_K4854007)[5]
gene. It is meant to express a fusion protein of the E. coli membrane protein CsgA and the barnacle cement protein Aacp19k.
Barnacle cement protein Aacp19k is an adhesive protein from the striped barnacle (Amphibalanus amphitrite). 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.
Cloning result
We successfully inserted CsgA+Aacp19k 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.
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+Aacp19k 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+Aacp19k did not had obvious adhesion to stick on the slides.
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+Aacp19k divided by the viscosity of J04450 (control), CsgA+Aacp19k was lower than 0.69%. We could tell that it was not really adhesive.
3. Modified ELISA
With the principle of ELISA antibody and antigen binding, we designed the modified ELISA to test whether CsgA+Aacp19k 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+Aacp19k was 2.94% lower than control (J04450). We could tell that the barnacle adhesive recombinant protein CsgA+Aacp19k could not capture antibodies effectively.
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_K4854007
[6] Liang Chao, Strickland Jack, Ye Zonghuang, Wu Wenjian, Hu Biru, Rittschof Dan, Biochemistry of Barnacle Adhesion: An Updated Review, Frontiers in Marine Science, Volume 6, 2019, URL: https://www.frontiersin.org/articles/10.3389/fmars.2019.00565
[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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 710
Illegal AgeI site found at 813 - 1000COMPATIBLE WITH RFC[1000]
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