Results | NYCU-Formosa

Overview

To establish our adhesive platform CoPlat, we constructed three categories of genes to express natural adhesive recombinant proteins, potential adhesive recombinant proteins, and functional adhesive recombinant proteins.
We performed flushing test, viscosity test, modified ELISA, and fluorescence test to detect the functions of proteins. The results showed that we succeeded in predicting the potential adhesive proteins. Moreover, CoPlat has great adhesive properties as expected, which can be used as an adhesive platform.

CoPlat Constructed by Natural Adhesive Recombinant Proteins

(1) Biobrick design

Our sequences of natural adhesive recombinant proteins were organized by CsgA recombinant with either mussel or barnacle proteins.

mfp3 — Figure 1. Biobrick design of mussel recombinant proteins.

mfp5 — Figure 1. Biobrick design of mussel recombinant proteins.

Bamcp20k-1 — Figure 2. Biobrick design of barnacle recombinant proteins.

cp19k — Figure 2. Biobrick design of barnacle recombinant proteins.

(2) Cloning

We successfully amplified our plasmid in E. coli DH5α and transformed into E. coli C41 for expression.

Figure 3. The digest check result of mussel recombinant proteins plasmids extracted from *E. coli* DH5α. M - Marker. 1 –backbone plasmid (pSB1A3, 2157 bp). 2 –backbone plasmid digest (pSB1A3, 2116 bp). 3 –insert (CsgA+Mfp3, 2945 bp). 4 –insert digest (CsgA+Mfp3, 829 bp). 5 –insert (CsgA+Mfp5, 3098 bp). 6 –insert digest (CsgA+Mfp5, 982 bp).

Figure 4. The digest check result of barnacle recombinant proteins plasmids extracted from *E. coli* DH5α. M - Marker. 1 –backbone plasmid (pSB1A3, 2157 bp). 2 –backbone plasmid digest (pSB1A3, 2116 bp). 3 –insert (CsgA+Bamcp20k-1, 3191 bp). 4 –insert digest (CsgA+Bamcp20k-1, 1075 bp). 5 –insert (CsgA+cp19k, 3323 bp). 6 –insert digest (CsgA+cp19k, 1207 bp). 7 –insert (CsgA+Mrcp20k, 3410 bp). 8 –insert digest (CsgA+Mrcp20k, 1294 bp). 9 –insert (CsgA+Aacp19k, 3413 bp). 10 –insert digest (CsgA+Aacp19k, 1297 bp).

(3) Functional test

a. Flushing test
With the flushing test, we can determine whether the natural adhesive recombinant proteins were adhesive initially.

Figure 5. The flushing test results of natural adhesive recombinant proteins.

We used backbone pSB3K3+J04450 as the control. From Figure 5, we can make a preliminary decision that CsgA+Mfp5, CsgA+cp19k, CsgA+Bamcp20k-1, CsgA+Mrcp20k had obvious adhesion to stick on the slides.

b. Viscosity test
In Prof. Ming-Chia, Lee's lab, we used rheometer to further test the viscosity of the proteins.

Figure 6. 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).

We used our backbone pSB3K3+J04450 as control. Taking the percentage of the viscosity of the natural adhesive recombinant proteins divided by the viscosity of J04450, CsgA+Mfp3 was 2.54% higher than control, CsgA+Mfp5 was 0.81% higher, CsgA+cp19k was 68.30% higher, CsgA+Aacp19k was less than 0.69%, CsgA+Bamcp20k-1 was 119.78% higher and CsgA+Mrcp20k was 120.44% higher. We could tell that three of our natural adhesive recombinant proteins were highly adhesive.
CsgA+Mfp3, CsgA+Mfp5, CsgA+cp19k, CsgA+Bamcp20k-1, and CsgA+Mrcp20k were respectively 2.54%, 0.81%, 68.30%, 119.78%, and 120.44% higher than the control, but CsgA+Aacp19k was 0.69% lower.

c. modified ELISA:
With the principle of ELISA antibody and antigen binding, we designed the modified ELISA to test whether natural adhesive recombinant proteins had a great ability to capture antibodies. We replaced the antigen with the produced protein, used the viscosity of the proteins to capture the antigen, and determined the strength of the binding antibody signal by OD₆₃₀. We used backbone pSB3K3+J04450 as the control, did a triple repeat, and took the average value as the data.

Figure 7. The modified ELISA test triple repeat OD₆₃₀ mean value of the 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.

Based on Figure 7, the ability of capturing the antibodies, CsgA+cp19k was 40.82 % better than J04450, CsgA+Bamcp20k-1 was 45.10% better and CsgA+Mrcp20k was 70.00% better. The barnacle recombinant proteins CsgA+cp19k, CsgA+Bamcp20k-1, and CsgA+Mrcp20k could capture antibodies effectively.

CoPlat Constructed by Potential Adhesive Recombinant Proteins.

(1) Biobrick design

We linked CsgA and the predicted four potential adhesive proteins together to create our CoPlat.

Figure 8. Biobrick design of potential adhesive recombinant proteins.

(2) Cloning

We successfully amplified our plasmid in E. coli DH5α and transformed into E. coli C41 for expression.

Figure 9. The digest check result of potential adhesive recombinant proteins plasmida extracted from *E. coli* DH5α. M –Marker. 1 –backbone plasmid (pSB1A3, 2157 bp). 2 –backbone plasmid digest (pSB1A3, 2116 bp). 3 –insert (CsgA+ecpA, 3278 bp). 4 –insert digest (CsgA+ecpA, 1162 bp). 5 –insert (CsgA+Nid1, 3776 bp). 6 –insert digest (CsgA+Nid1, 1660 bp). 7 –insert (CsgA+epd2, 3464 bp). 8 –insert digest (CsgA+epd2, 1348 bp).

Figure 10. The digest check result of potential adhesive recombinant proteins plasmids extracted from *E. coli* DH5α. M –Marker. 1 –backbone plasmid (pSB1A3, 2157 bp). 2 –backbone plasmid digest (pSB1A3, 2116 bp). 3 –insert (CsgA+zig-4, 3560 bp). 4 –insert digest (CsgA+zig-4, 1444 bp).

(3) Functional test

a. Flushing test
With the flushing test, we can determine whether the potential adhesive recombinant proteins were adhesive initially.

Figure 11. The flushing test results of potential adhesive recombinant proteins.

With the flushing test, we can see that all four of our potential adhesive recombinant proteins had adhesion and were able to stick tightly to the slides.

b. Viscosity test
In Prof. Ming-Chia, Lee's lab, we used rheometer to further test the viscosity of potential adhesive recombinant proteins.

Figure 12. The viscosity test results of potential adhesive recombinant proteins compared with barnacle recombinant proteins CsgA+Mrcp20k. The y-axis is the percentage of the viscosity of the adhesive recombinant proteins divided by the viscosity of J04450 (control).

With the same process, we could know that potential adhesive recombinant protein CsgA+ecpA was 39.02% higher than the control J04450, CsgA+Nid1 was 44.04% higher, CsgA+epd2 was 109.60% higher, and CsgA+zig-4 was 314.15% higher.
Hence, it can be proved that all of our potential adhesive recombinant proteins have significant viscosity, especially CsgA+zig-4.

c. modified ELISA:
We performed modified ELISA to test whether the potential adhesive recombinant proteins had a great ability to capture antibodies.

Figure 13. 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.

With the same process, we can know that CsgA+ecpA is 4.88% better than backbone J04450, CsgA+Nid1 is 2.44% better, CsgA+epd2 is 2.44% lower and CsgA+zig-4 is 114.63% better.
CsgA+zig-4 were adhesive and had a great ability to capture the antibody at the same time.

	CsgA+ecpA	CsgA+Nid1	CsgA+epd2	CsgA+zig-4
Rheometer	39.02%	44.04%	109.60%	314.15%
ELISA	4.88%	2.44%	-2.44%	114.63%

Table 1. The percentage of potential adhesive recombinant proteins comparison with J04450 (control).

4. CoPlat Constructed with Functional Recombinant

(1) Biobrick design

To make our CoPlat more specific, we used GFP as functional protein. Then recombine GFP, CsgA and Mfp5 to create the CoPlat.

Figure 14. Biobrick design of functional adhesive recombinant protein.

(2) Cloning

We successfully amplified our plasmid in E. coli DH5α and transformed into E. coli C41 for expression.

Figure 15. The digest check result of functional adhesive recombinant protein plasmid extracted from *E. coli* DH5α. M –Marker. 1 –backbone plasmid (pSB1A3, 2157 bp). 2 –backbone plasmid digest (pSB1A3, 2116 bp). 3 –insert (GFP+CsgA+Mfp5, 3842 bp). 4 –insert digest (GFP+CsgA+Mfp5, 1726 bp).

(3) Functional test

a. Flushing test
With the flushing test, we can determine whether the functional adhesive recombinant protein was adhesive initially.

Figure 16. The flushing test result of functional adhesive recombinant protein.

In contrast to CsgA+Mfp5 and GFP, our functional adhesive recombinant protein had better adhesion. It could stick to slide marvelously.

b. Viscosity test
In Prof. Ming-Chia, Lee's lab, we used rheometer to further test the viscosity of functional adhesive recombinant protein.

Figure 17. The viscosity test results of adhesive recombinant proteins. The y-axis is the percentage of the viscosity of the control and GFP+CsgA+Mfp5 divided by the viscosity of J04450 (control).

According to Figure 17, we can see that functional adhesive recombinant protein was 338.92% more adhesive than the control J04450, 404.91% more adhesive than mussel recombinant protein and 187.77% more adhesive than GFP. Functional adhesive recombinant protein is extremely adhesive.

c. modified ELISA:
We performed modified ELISA to test whether the Functional adhesive recombinant protein had ability to capture antibodies.

Figure 18. 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.

By Figure 18, we could tell that the functional adhesive recombinant protein had a better ability to capture antibodies than J04450, CsgA+Mfp5 and GFP obviously.

d. 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.

Figure 19. The fluorescent bacteria comparison between GFP and functional adhesive recombinant protein.

According to Figure 19, GFP+CsgA+Mfp5 was adhesive while maintaining its functionality.

Summary

Comprehensive the results of the three functional tests, First, the barnacle recombinant proteins CsgA+cp19k, CsgA+Bamcp20k-1, and CsgA+Mrcp20k had both adhesion and the ability to capture antibodies. Second, all of our predicted potential adhesive recombinant proteins were adhesive, and CsgA+zig-4 not only had the highest adhesion but also captured the antibody most effectively. In addition, our functional protein had terrifically adhesion and could capture the antibody tightly. At the same time, it can remain the function of the linked functional protein.
We sufficiently succeeded in predicting potential adhesive proteins with adhesive properties. Moreover, the results of three functional tests confirmed that our CoPlat idea can be practically implemented, whether it is non-specific CoPlat or specific CoPlat with functional proteins attached by linkers.