iGEM 2023 — Team nycu-formosa

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Notebook

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Cycle 1
Cycle 2
Cycle 3
Cycle 4

## Cycle 1

#### **Design**

To create an adhesive platform called CoPlat, we selected six adhesive proteins as the source of adhesion.
These proteins consist of natural adhesive proteins, such as mussel protein (Mfp3, Mfp5), and barnacle
protein (Bamcp20K-1, cp19K, Mrcp20K, Aacp19K). We connected each of these adhesive proteins with a GS
linker to the membrane protein gene CsgA, allowing the adhesive proteins to anchor on the cell membrane of
Escherichia coli and create CoPlat. Finally, we added the lac promoter for future protein expression. To
prove the following three points, we designed several experiments:

1. Whether the adhesive proteins form on the membrane.

2. Whether CoPlat can maintain adhesion and compare the adhesion of different adhesive proteins to select
the most suitable CoPlat adhesive protein.

3. Whether the adhesive proteins have the ability to capture antibodies or enzymes and similar substances.

Establishing an adhesive standard that can serve as a reference for whether a protein possesses adhesion.

![BioBrick of our CoPlat](https://static.igem.wiki/teams/4854/wiki/wiki-picture/latest-biobricks/adhesive-2.png)

Figure 1. Biobrick design of natural adhesive recombinant proteins

---

#### **Build**

###### 1. Cloning result

![cloning result-1](https://static.igem.wiki/teams/4854/wiki/wiki-picture/final-cycle/fig-2.png)

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

![cloning result-2](https://static.igem.wiki/teams/4854/wiki/wiki-picture/final-cycle/fig-3.png)

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

---

###### 2. SDS result

After cloning, we induced the bacteria with 200 μM IPTG for 12 hours to express the proteins. However,
when performing SDS-PAGE, we did not observe protein expression. After extensive research and consulting
with experts, we speculated that this could be due to the likelihood of membrane proteins having low
expression levels, making it difficult to detect whether the target protein has been expressed through
SDS-PAGE.

![SDS-PAGE result](https://static.igem.wiki/teams/4854/wiki/wiki-picture/final-cycle/fig-4.png)

Figure 4. The SDS page result of mussel adhesive recombinant protein (CsgA + Mfp5) from *E.
coli* BL21. M - Marker. 1 – backbone plasmid (pSB1A3). 2 – insert (CsgA+Mfp5-2, 25.02 kDa). 3 –
insert (CsgA+Mfp5-8, 25.02 kDa).

Since SDS-PAGE could not confirm protein expression, we decided to directly confirm the production of the
target protein through functional assays. In all of our tests, we assumed that the target gene could reach
the cell membrane, and none of our tests resulted in cell disruption.

---

#### **Test**

###### 1. Flushing test

To confirm that CoPlat can adhere to a flat surface, we spread bacterial liquid onto glass slides and
cultured it for 30 minutes, allowing the bacteria to attach using adhesive proteins. We then rinsed the
slides and compared the results of slides with and without adhesive proteins under a microscope.

![flushing test result of adhesive protein](https://static.igem.wiki/teams/4854/wiki/wiki-picture/flushing-test-final-result/natural.png)

Figure 5. The flushing test result of adhesive proteins

We observed that in the presence of bacteria with adhesive proteins, there were more residual bacteria on
the slides, demonstrating the effectiveness of adhesive proteins, with the Mrcp20k sequence having the
highest adhesion.

---

###### 2. Viscosity test

In order to recheck whether the CoPlat possesses adhesion, we used a rheometer to measure the shear forces
of different proteins and attempted to quantitatively assess the adhesion of each adhesive protein based
on the results. Additionally, we analyzed which adhesive protein exhibited the highest adhesion strength.

A. First Test: Except for Mrcp20k, no comparison of adhesion of adhesive proteins was observed, and we
suspected that this was due to not switching the vector to a plasmid with higher expression capability,
resulting in lower protein expression. Therefore, we replaced the vector from the originally lower
expression pSB1A3 with pSB3K3 and conducted the viscosity test again.
![rheometer Test Result vs. BL21](https://static.igem.wiki/teams/4854/wiki/wiki-picture/final-cycle/fig-6.jpg)

Figure 6. The viscosity test 1 results of adhesive recombinant proteins. Y-axis is the
percentage of adhesive proteins viscosity/ J04450 (control) viscosity.

---

B. Second Test: It was observed that bacteria with adhesive proteins exhibited higher adhesion compared to
the control group(pSB3K3-J04450), with cp19k, Bamcp20k-1, and Mrcp20k possess adhesion.

![viscosity test result vs. J04450](https://static.igem.wiki/teams/4854/wiki/wiki-picture/final-cycle/fig-7.png)

Figure 7. The viscosity test 2 results of natural adhesive recombinant proteins. Y-axis is the
percentage of adhesive proteins viscosity/ J04450 (control) viscosity

---

###### 3. Modified ELISA

Furthermore, we wanted to prove our CoPlat have ability to capture antibodies or related substances, we
tried modifying the ELISA test. We attempted to culture labeled antibodies with CoPlat for 30 minutes
and
added TMB, allowing adhesive proteins that successfully captured antibodies to show color.

A. First Test: To prevent the blocking buffer from being stuck by adhesive proteins, which would hinder
successful TMB color development, we omitted the blocking step and used the Eppendorf method for
testing.

However, we found that the pure PB solution without any adhesive proteins showed brighter than the other
groups, which was contrary to our initial assumption. We suspected that the antibodies adhered to the
walls of the Eppendorf tube during the incubation period, causing TMB coloration.

![viscosity test result vs. J04450](https://static.igem.wiki/teams/4854/wiki/wiki-picture/final-cycle/fig-8.jpg)

Figure 8. The Modified ELISA Test 1 triple repeat OD630 mean value of adhesive
recombinant proteins. The Y-axis is the percentage of adhesive proteins OD630 mean value /
J04450 (control) OD630 mean value.

B. Second Test: To improve the experiment, we transferred the Modified ELISA test to a new Eppendorf
tube
after removing the antibodies and conducted the test again.

![ELISA triple repeat OD](https://static.igem.wiki/teams/4854/wiki/wiki-picture/final-cycle/fig-9.png)

Figure 9. The Modified ELISA Test 2 triple repeat OD630 mean value of nature
adhesive recombinant proteins. Y-axis is the percentage of adhesive proteins OD630 mean
value
/ J04450 (control) OD630 mean value

From the coloration results, it can be seen that E. coli with adhesive protein genes can capture labeled
antibodies, allowing TMB to show color. In this graph, we can infer that CoPlat with cp19k, Bamcp20k-1,
and
Mrcp20k have adhesion. This result corresponds to the rheometer test, which indicates that the ability
to
capture antibodies may relate to the adhesion strength from adhesive protein of CoPlat.

---

#### **Learn**

1. Since all our tests do not conduct cell disruption, yet the function remained normal, it can be
inferred that the natural adhesive recombinant proteins are successfully produced and correctly anchor on
the membrane of *E.coli*.

2. Based on the results from test1~3, cp19k, Bamcp20k-1, Mrcp20k exhibit adhesion, with Mrcp20k showing
the best performance.

3. From the results of the Modified ELISA test, it is evident that our adhesive proteins can immobilize
antibodies or related substances.

4. We established an adhesion standard that can be used to analyze whether the sample has adhesion.

## Cycle 2

#### **Design**

We are looking for the possibility to get a better adhesive protein, that is stronger, more adhesive, and
easier to produce, so we made an attempt to design a classifier that can find proteins with general
adhesive, but its adhesion is not recorded in the protein databases.

---

#### **Build**

To find the potentially adhesive proteins, we built a classifier based on a long short-term memory
network(LSTM) with one hot encoding and trained it with the training data we mentioned above.

---

#### **Test**

The model reached a poor accuracy of 0.4.

---

#### **Learn**

We realized that LSTM does not work well with insufficient training data. Therefore, we gave up on the
deep learning methods and wanted to build the classifier with a simpler machine learning model.

## Cycle 3

#### **Design**

To make our classifier more reliable, we delved into the ESM encoding method to determine how to best
represent protein sequences effectively. In the previous cycle, we realized that the quantity of training
data collected was insufficient to support a deep learning model such as LSTM. As a result, we selected
support vector machine as our machine learning method.

![potential protein biobricks](https://static.igem.wiki/teams/4854/wiki/wiki-picture/latest-biobricks/potential-adhesive-2.png)

Figure 10. Biobrick design of potential adhesive recombinant proteins

To validate the predicted potential adhesive proteins and compare their adhesion with existing adhesive
proteins, we designed biobricks similar to Cycle 1. Specifically, we connected each of the predicted four
potential adhesive proteins (Nid1, ecpA, epd2, zig-4) to the membrane protein gene CsgA using GS linkers,
allowing them to form on the cell membrane of Escherichia coli to create a CoPlat based on the adhesive
proteins. Finally, we added the lac promoter for future protein expression.

Since we established adhesive protein standards in Cycle 1, we attempted to compare the adhesion and
functional tests with existing adhesive proteins. We aimed to determine whether our predicted potential
adhesive proteins were correct. Therefore, we designed functional tests similar to those in Cycle 1.

#### **Build**

Once we have a well-defined problem and the dataset in place, we will transition to the build phase. Here,
we will focus on data preprocessing, ensuring that the dataset is clean, any missing values are handled
appropriately, and the ESM-encoded features are processed as needed, such as normalization or scaling.
Next, we will proceed with the implementation of the SVM classifier using a suitable framework,
integrating the ESM encoding to convert protein sequences into feature vectors. Training the model will
involve splitting the dataset into training and validation sets, fine-tuning hyperparameters, and closely
monitoring the model's performance.

![potential digest check result](https://static.igem.wiki/teams/4854/wiki/wiki-picture/final-cycle/fig-11.png)

Figure 11. The digest check result of potential adhesive recombinant proteins plasmid
extracted from *E. coli* DH5α. M - Marker. 1 – backbone plasmid (pSB1A3, 2157bp). 2 – backbone
plasmid digest (pSB1A3, 2116bp). 3 – insert (CsgA+ecpA, 3278bp). 4 – insert digest (CsgA+ecpA, 1162bp).
5 – insert (CsgA+Nid1, 3776bp). 6 – insert digest (CsgA+Nidi, 1660bp). 7 – insert (CsgA+zig-4, 3464bp).
8 – insert digest (CsgA+zig-4, 1348bp).

![potential digest check result](https://static.igem.wiki/teams/4854/wiki/wiki-picture/final-cycle/fig-12.png)

Figure 12. The digest check result of potential adhesive recombinant proteins plasmid
extracted from *E. coli* DH5α. M - Marker. 1 – backbone plasmid (pSB1A3, 2157bp). 2 – backbone
plasmid digest (pSB1A3, 2116bp). 3 – insert (CsgA+zig-4, 3560bp). 4 – insert digest (CsgA+zig-4,
1444bp).

#### Test

###### 1. Flushing Test

To confirm that CoPlat, based on potential adhesive proteins, can adhere to a flat surface, we spread
bacterial liquid onto glass slides and cultured it for 30 minutes, allowing the bacteria to attach using
potential adhesive proteins. We then rinsed the slides and compared the results of slides under a
microscope with and without our predicted adhesive proteins.

![Flusing Test of potential adhesive protein](https://static.igem.wiki/teams/4854/wiki/wiki-picture/flushing-test-final-result/potential.png)

Figure 13. The flushing test result of potential adhesive recombinant proteins

We observed that there were more residual bacteria on the slides in the presence of bacteria with adhesive
proteins, demonstrating that potential adhesive proteins have adhesion comparable to existing proteins.

---

###### 2. Viscosity test

In order to recheck whether the CoPlat possesses adhesion, we used a rheometer to quantitatively measure the
shear forces of potential adhesive proteins. Additionally, we analyzed which potential adhesive protein
exhibited the highest adhesion strength.

![Rheometer test of potential adhesive protein](https://static.igem.wiki/teams/4854/wiki/wiki-picture/final-cycle/fig-14.png)

Figure 14. The viscosity test results of potential adhesive recombinant proteins comparison with
barnacle adhesive recombinant proteins CsgA+Mrcp20k. Y-axis is the percentage of adhesive proteins
viscosity/ J04450 (control) viscosity.

We observed that bacteria with potential adhesive proteins exhibited higher adhesion compared to the control
group, with adhesion strength in the following order: zig-4 > epd2 > Nid1 > ecpA.

---

###### 3. Modified ELISA

Furthermore, we wanted to prove our CoPlat have ability to capture antibodies or related substances, we
tried modifying the ELISA test. We attempted to culture labeled antibodies with potential adhesive proteins
for 30 minutes and added TMB, allowing adhesive proteins that successfully captured antibodies to produce
color.

![Rheometer Test of potential adhesive protein](https://static.igem.wiki/teams/4854/wiki/wiki-picture/final-cycle/fig-15.png)

Figure 15. The Modified ELISA Test triple repeat OD630 mean value of potential
adhesive recombinant proteins. The Y-axis is the percentage of adhesive proteins OD630 mean
value / J04450 (control) OD630 mean value.

From the results, it can be inferred that zig-4 has adhesion, similar to existing adhesive proteins.
However, the other predicted adhesive proteins cannot be verified whether they have adhesion through the
modified ELISA test results.

#### **Learn**

1. Since all our tests do not conduct cell disruption, yet the function remained normal, it can be
inferred that the potential adhesive recombinant proteins are successfully produced and correctly anchor
on the membrane of *E. coli*.

2. From the test 1~3 results, it is the evident that potential adhesive proteins possess adhesion, with
adhesion strength in the following order: zig4 > epd2 > Nid1 > ecpA.

3. From the results of the Modified ELISA test, it is the evident that our potential adhesive proteins can
immobilize antibodies or related substances.

## Cycle 4

#### **Design**

After confirming that CoPlat can be expressed on the membrane, we attempted to link functional proteins to
CoPlat, giving it not only adhesion but also the ability to express additional functions. Therefore, we
used a “10 amino acid flexible protein domain linker” to connect our functional protein to the CoPlat
designed in Cycle 1. The reason we did not use GS linker was to avoid excessively repetitive sequences,
which could complicate sequence synthesis. Additionally, we linked our functional protein before and after
CoPlat, and we attempted to verify which arrangement forms a more stable structure and exhibits better
functionality. We used GFP as a verification method for our functional protein. If GFP can successfully
express green fluorescence, it verifies that the functional protein is functioning normally on CoPlat. To
prove the following three points, we designed several experiments for confirmation:

1. The functional protein can successfully link with CoPlat and be expressed on the cell membrane.

2. It can maintain a certain level of adhesion.

3. The functional protein can function normally.

4. Which arrangement forms a more stable structure and exhibits better functionality.

(In the following text, M5G and GM5 represent the functional protein linked to CoPlat.)

![Biobrick for potential adhesive protein](https://static.igem.wiki/teams/4854/wiki/wiki-picture/latest-biobricks/gm5-2.png)

Figure 16. Biobrick design of functional recombinant protein M5G

![Biobrick for potential adhesive protein](https://static.igem.wiki/teams/4854/wiki/wiki-picture/latest-biobricks/m5g-2.png)

Figure 17. Biobrick design of functional recombinant protein GM5

---

#### **Build**

Cloning result: While performing the cloning steps, we consistently observed deletions in M5G during
sequencing. This unsuccessful cloning of M5G can be attributed to the gene mutation, which also proves
that GM5 have more stable structure than M5G.

![digest check graph](https://static.igem.wiki/teams/4854/wiki/wiki-picture/final-cycle/fig-18.png)

Figure 18. The digest check result of functional recombinant protein plasmid extracted from
*E. coli* DH5α. M - Marker. 1 –backbone plasmid (pSB1A3, 2157bp). 2 –backbone plasmid digest
(pSB1A3, 2116bp). 3 –insert (GFP+CsgA+Mfp5, 3842bp). 4 –insert digest (GFP+CsgA+Mfp5, 1726bp).

#### Test

###### 1. Flushing Test

To confirm that CoPlat can adhere to a flat surface after adding the functional protein, we spread
bacterial liquid onto glass slides and cultured it for 30 minutes, allowing the bacteria to attach. We
then rinsed the slides and compared the results of slides under a microscope with and without GM5.

![Flusing Test of potential adhesive protein](https://static.igem.wiki/teams/4854/wiki/wiki-picture/flushing-test-final-result/gm5.png)

Figure 19. The flushing test result of functional recombinant protein

We observed that in the presence of bacteria with GM5, there were more residual bacteria on the slides,
demonstrating that CoPlat can still maintain adhesion after adding the functional protein.

---

###### 2. Viscosity Test

In order to recheck whether the CoPlat linked to the functional protein possesses adhesion, we used a
rheometer to measure the shear forces of different proteins. We attempted to quantify the adhesion of each
adhesive protein based on the results and compared it with existing adhesive proteins to determine if the
adhesion matches.

![ELISA test of potential adhesive protein](https://static.igem.wiki/teams/4854/wiki/wiki-picture/final-cycle/fig-20.png)

Figure 20. The viscosity test results of functional recombinant protein. The Y-axis is the
percentage of adhesive proteins viscosity/ J04450 (control) viscosity.

It can be observed that CoPlat with GM5 exhibited higher adhesion compared to the control group,
confirming that CoPlat can still maintain adhesion after adding the functional protein.

---

###### 3. Fluorescence Test

By comparing the green fluorescence intensity at the 545 nm wavelength between pure GFP and GM5, we
determined whether the functional protein's activity is reduced due to its connection to CoPlat.

![Rheometer Test of potential adhesive protein](https://static.igem.wiki/teams/4854/wiki/wiki-picture/for-igem-part/flushing-test/gm5-flourence.png)

Figure 21. The flushing test result of functional recombinant protein (fluorescence graph)

![Rheometer Test of potential adhesive protein](https://static.igem.wiki/teams/4854/wiki/wiki-picture/final-cycle/fig-22.jpg)

Figure 22. Fluorescence Test results of functional recombinant protein. The Y-axis is the
fluorescence intensity of GFP (OD*475*) /empty C41 strain (control).

From the data, it is the evident that GM5 has normally expressed the GFP gene. However, its fluorescence
intensity is lower than that of the pure GFP strain, indicating that CoPlat still slightly affects the
functional protein's functionality.

#### **Learn**

1. From the viscosity and flushing test results, it can be concluded that CoPlat, when connected to the
functional protein, can still express itself on the membrane normally and does not affect the inherent
adhesion of CoPlat.

2. From the fluorescence test results, it is evident that CoPlat allows the functional protein to maintain
its functionality normal when linked to the functional protein using the linker.

3. The functional protein linked before CoPlat forms a much more stable structure, which will be the final
arrangement of our CoPlat.

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## ![Protocol-cloning](https://static.igem.wiki/teams/4854/wiki/wiki-picture/exp-protocol-icon/cloning.png)

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Protocol-cloning

## ![Protocol-expression-ft](https://static.igem.wiki/teams/4854/wiki/wiki-picture/exp-protocol-icon/expression-ft.png)

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Protocol-Protein expressioing and functional test

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## 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](https://static.igem.wiki/teams/4854/wiki/wiki-picture/latest-biobricks/mfp3-2.png)
![mfp5](https://static.igem.wiki/teams/4854/wiki/wiki-picture/latest-biobricks/mfp5-2.png)

Figure 1. Biobrick design of mussel recombinant proteins.

---

![Bamcp20k-1](https://static.igem.wiki/teams/4854/wiki/wiki-picture/latest-biobricks/bamcp20k-1-2.png)
![cp19k](https://static.igem.wiki/teams/4854/wiki/wiki-picture/latest-biobricks/cp19k-2.png)
![Mrcp20k](https://static.igem.wiki/teams/4854/wiki/wiki-picture/latest-biobricks/mrcp20k-2.png)
![Aacp19k](https://static.igem.wiki/teams/4854/wiki/wiki-picture/latest-biobricks/aacp19k-2.png)

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.

![](https://static.igem.wiki/teams/4854/wiki/wiki-picture/result-final-fig/fig3.png)

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).

![](https://static.igem.wiki/teams/4854/wiki/wiki-picture/result-final-fig/fig4.png)

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.

![](https://static.igem.wiki/teams/4854/wiki/wiki-picture/result-final-fig/fig5.png)

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.

![](https://static.igem.wiki/teams/4854/wiki/wiki-picture/result-final-fig/fig6.png)

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.

![](https://static.igem.wiki/teams/4854/wiki/wiki-picture/result-final-fig/fig7.png)

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.

![](https://static.igem.wiki/teams/4854/wiki/wiki-picture/latest-biobricks/ecpa-2.png)
![](https://static.igem.wiki/teams/4854/wiki/wiki-picture/latest-biobricks/nid1-2.png)
![](https://static.igem.wiki/teams/4854/wiki/wiki-picture/latest-biobricks/epd2-2.png)
![](https://static.igem.wiki/teams/4854/wiki/wiki-picture/latest-biobricks/zig-4-2.png)

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.

![](https://static.igem.wiki/teams/4854/wiki/wiki-picture/result-final-fig/fig9.png)

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).

![](https://static.igem.wiki/teams/4854/wiki/wiki-picture/result-final-fig/fig10.png)

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.

![](https://static.igem.wiki/teams/4854/wiki/wiki-picture/result-final-fig/fig11.png)

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.

![](https://static.igem.wiki/teams/4854/wiki/wiki-picture/result-final-fig/fig12.png)

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.

![](https://static.igem.wiki/teams/4854/wiki/wiki-picture/result-final-fig/fig13.png)

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.

![](https://static.igem.wiki/teams/4854/wiki/wiki-picture/latest-biobricks/gm5-2.png)

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.

![](https://static.igem.wiki/teams/4854/wiki/wiki-picture/result-final-fig/fig15.png)

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.

![](https://static.igem.wiki/teams/4854/wiki/wiki-picture/result-final-fig/fig16.png)

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.

![](https://static.igem.wiki/teams/4854/wiki/wiki-picture/result-final-fig/fig17.png)

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.

![](https://static.igem.wiki/teams/4854/wiki/wiki-picture/result-final-fig/fig18.png)

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.

![](https://static.igem.wiki/teams/4854/wiki/wiki-picture/result-final-fig/fig19.png)

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.

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