iGEM 2020 — Team PuiChing_Macau

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# Team:PuiChing Macau/Notebook

[![iGEM PuiChing Macau](https://static.igem.org/mediawiki/2020/d/d2/T--Puiching_Macau--logo.png)](https://2020.igem.org/Team%3APuiChing_Macau)

* Project

  + [Description](https://2020.igem.org/Team%3APuiChing_Macau/Description)
  + [Design](https://2020.igem.org/Team%3APuiChing_Macau/Design)
  + [Engineering Success](https://2020.igem.org/Team%3APuiChing_Macau/Engineering)
  + [Results & Contributions](https://2020.igem.org/Team%3APuiChing_Macau/Contribution)
  + [Model](https://2020.igem.org/Team%3APuiChing_Macau/Model)
  + [Implementation](https://2020.igem.org/Team%3APuiChing_Macau/Implementation)
  + [Proof of Concept](https://2020.igem.org/Team%3APuiChing_Macau/Proof_Of_Concept)
* Outreach

  + [Human Practices & Integrated HP](https://2020.igem.org/Team%3APuiChing_Macau/Human_Practices)
  + [Collaborations](https://2020.igem.org/Team%3APuiChing_Macau/Collaborations)
  + [Education](https://2020.igem.org/Team%3APuiChing_Macau/Education)
* Lab

  + [Parts](https://2020.igem.org/Team%3APuiChing_Macau/Parts)
  + [Notebook](https://2020.igem.org/Team%3APuiChing_Macau/Notebook)
  + [Safety](https://2020.igem.org/Team%3APuiChing_Macau/Safety)
  + [Protocol](https://2020.igem.org/Team%3APuiChing_Macau/Protocol)
* About Us

  + [Team](https://2020.igem.org/Team%3APuiChing_Macau/Team)
  + [Attributions](https://2020.igem.org/Team%3APuiChing_Macau/Attributions)
* [Judging Form](https://igem.org/2020_Judging_Form?id=3503)

![](https://static.igem.org/mediawiki/2020/thumb/6/63/T--PuiChing_Macau--Decor--1.png/800px-T--PuiChing_Macau--Decor--1.png)

Notebook

## Our Notebook

This is our notebook below, data collected throughout the whole year...

**⭐ Please click the buttons below to show detailed information ⭐**

Week 1
Week 2
Week 3
Week 4
Week 5
Week 6
Week 7

## Primer design and experiment preparation

#### 1) Design and order primers for later on DNA extraction with products having the desired overhang/extra DNA 2) Culture medium preparation for later on use in transformation

## 1)Building and confirming construct

#### 1)Amplify and extract fragments of K3503001 (alpha casein) K1608000 (SR Protein) K1321014(cellulose binding domain), K3089023(mussel adhesion protein)

a) Confirmation with Gel electrophoresis
b) DNA purification (Gel extraction) of the correct DNA fragments from gel

#### 2) Link pET with different overhangs : pET11a-Ptac-F, pET11a

a) Confirmation with Gel electrophoresis

#### 3) The DNA fragments were then ligated to pET form three constructs Alpha casein(K3503001), CBD-Alpha casein(K3503008), mfp5-Alpha casein(K3503007), mfp5-SR(K3503006), CBD-SR(K3503004), ptac-SR-GST(K1608000)

a) DNA extraction with miniprep kit and confirm with PCR and gel electrophoresis then undergo transformation to E.coli.

#### 4) Optimizing Restriction Endonuclease Reactions for (mfp5-SR), (mfp5-Alpha casein), (CBD- Alpha casein), (ptac-SR)

![](https://static.igem.org/mediawiki/2020/b/b6/T--Puiching_Macau--Agarose_gel_electrophoresis.png)

* Lane 1: Invitrogen trackit 1Kb plus DNA Ladder.
* Lane 2:pET-11a-mfp5-SR(K3503006)
* Lane 3:pET-11a-mfp5 alpha casein (K3503007)
* Lane 4: pET-11a-ptac GST-SR (K1608000)
* Lane 5:pET11a

#### 1)Protein extraction (Alpha casein)

#### 2)Sonication: use microtip, set 40% AMPL, 45 secs processing, 1 sec on-off, sonicate for 3 times

#### 3)IPTG induction of Alpha casein(K3503001), mfp5-Alpha casein(k3503007), CBD-Alpha casein (K3503008)

(a)Incubation of 0.5h, 1h, 2h and 4h. Remove supernatant. Freeze pellet at -20 until needed

#### 4) Prepare for the SDS-Page Gel

#### 5)Coomassie Blue stain

a) Bio rad protein dual color ladder, 2) pet11a, 3-4) SR(K1608000) 5-6) K3503006(mfp5-SR) 7-8) K3503004(CBD-SR). Lane 4,6,8 are with 1mM IPTG induction.)

![](https://static.igem.org/mediawiki/2020/d/d5/T--Puiching_Macau--alpha_blue.png)

#### 6) Western blot

![](https://static.igem.org/mediawiki/2020/9/9d/T--Puiching_Macau--Alpha_western.png)

#### 1)Protein extraction SR protein

#### 2)Sonication: use microtip, set 40% AMPL, 45 secs processing, 1 sec on-off, sonicate for 3 times

#### 3)IPTG induction of SR protein(K1608000), mfp5-SR protein(k3503006), CBD-SR protein (K3503004)

(a)Incubation of 0.5h, 1h, 2h and 4h. Remove supernatant. Freeze pellet at -20 until needed.

#### 4) Prepare for the SDS-Page Gel

#### 5)Coomassie Blue stain

(Lane1) Bio-rad protein dual-color ladder, 2) pet11a, 3-4) K3503001(Alpha casein), 5-6) K3503007(mfp5-Alpha casein), 7-8) K3507008(CBD-Alpha casein). Lane 4,6,8 are with 0.1mM IPTG induction.)

![](https://static.igem.org/mediawiki/2020/9/9b/T--Puiching_Macau--SR_Blue.png)

#### 6) Western blot

![](https://static.igem.org/mediawiki/2020/8/85/T--Puiching_Macau--SR_western.png)

## Check the fluorescent on the fabrics

#### 1)We check the fluorescent on the fabrics with Nikon A1MP multiphoton confocal microscope and fluorescence confocal microscope.

![](https://static.igem.org/mediawiki/2020/8/8a/T--Puiching_Macau--Nikon_A1MP.png)

#### 2) We confirmed that the red fluorescence has an almost unobvious part

#### 3)We finally choose RFP (red fluorescence protein) to add on our protein to check the adhesive.

#### 1)Protein extraction (Alpha casein)

#### 2)Sonication: use microtip, set 40% AMPL, 45 secs processing, 1 sec on-off, sonicate for 3 times

#### 3)IPTG induction of Alpha casein(K3503001), mfp5-Alpha casein(k3503007), CBD-Alpha casein (K3503008)

(a)Incubation of 0.5h, 1h, 2h and 4h. Remove supernatant. Freeze pellet at -20 until needed.

#### 4) Prepare for the SDS-Page Gel

#### 5)Coomassie Blue stain

![](https://static.igem.org/mediawiki/2020/0/00/T--Puiching_Macau--RFP_blue.png)

(Lane 1)Bio rad protein dual color ladder, 2) pet11a, 3-4) SR(K1608000) 5-6) K3503006(mfp5-SR) 7-8) K3503004(CBD-SR). Lane 4,6,8 are with 1mM IPTG induction.)

### Adhesion test

Comparing with bedsheet after washing and soaking

![](https://static.igem.org/mediawiki/2020/6/66/T--Puiching_Macau--The_percentage.png)

The percentage of the protein after processes

### Fire testing

![](https://static.igem.org/mediawiki/2020/9/9c/T--Puiching_Macau--bedsheet_chart.png)

Vertical Burning Test using bedsheet

![](https://static.igem.org/mediawiki/2020/2/2e/T--Puiching_Macau--wood.png)

Fire retardant test for wood

### IR spectrum between water and protein-coated bed sheets

![](https://static.igem.org/mediawiki/2020/d/da/T--PuiChing_Macau--protein_ir.jpg)

IR spectrum of engineered flame retardant on bed sheet

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* Project
  + [Description](https://2020.igem.org/Team%3APuiChing_Macau/Description)
  + [Design](https://2020.igem.org/Team%3APuiChing_Macau/Design)
  + [Model](https://2020.igem.org/Team%3APuiChing_Macau/Model)
  + [Eng. Success](https://2020.igem.org/Team%3APuiChing_Macau/Engineering)
  + [Implementation](https://2020.igem.org/Team%3APuiChing_Macau/Implementation)
  + [Rslt. & Contrib.](https://2020.igem.org/Team%3APuiChing_Macau/Contribution)
  + [Prf. of Concept](https://2020.igem.org/Team%3APuiChing_Macau/Proof_Of_Concept)
* Outreach
  + [Human Practices & IHP](https://2020.igem.org/Team%3APuiChing_Macau/Human_Practices)
  + [Collaborations](https://2020.igem.org/Team%3APuiChing_Macau/Collaborations)
  + [Education](https://2020.igem.org/Team%3APuiChing_Macau/Education)
* Lab
  + [Parts](https://2020.igem.org/Team%3APuiChing_Macau/Parts)
  + [Notebook](https://2020.igem.org/Team%3APuiChing_Macau/Notebook)
  + [Safety](https://2020.igem.org/Team%3APuiChing_Macau/Safety)
  + [Protocol](https://2020.igem.org/Team%3APuiChing_Macau/Protocol)
* About Us
  + [Team](https://2020.igem.org/Team%3APuiChing_Macau/Team)
  + [Attributions](https://2020.igem.org/Team%3APuiChing_Macau/Attributions)
* [Judging Form](https://igem.org/2020_Judging_Form?id=3503)

* @iGEMPuiChing2020

* igem.wmc@g.puiching.edu.mo

© 2020 iGEM PuiChing Macau Media Team

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# Team:PuiChing Macau/Engineering

[![iGEM PuiChing Macau](https://static.igem.org/mediawiki/2020/d/d2/T--Puiching_Macau--logo.png)](https://2020.igem.org/Team%3APuiChing_Macau)

* Project

  + [Description](https://2020.igem.org/Team%3APuiChing_Macau/Description)
  + [Design](https://2020.igem.org/Team%3APuiChing_Macau/Design)
  + [Engineering Success](https://2020.igem.org/Team%3APuiChing_Macau/Engineering)
  + [Results & Contributions](https://2020.igem.org/Team%3APuiChing_Macau/Contribution)
  + [Model](https://2020.igem.org/Team%3APuiChing_Macau/Model)
  + [Implementation](https://2020.igem.org/Team%3APuiChing_Macau/Implementation)
  + [Proof of Concept](https://2020.igem.org/Team%3APuiChing_Macau/Proof_Of_Concept)
* Outreach

  + [Human Practices & Integrated HP](https://2020.igem.org/Team%3APuiChing_Macau/Human_Practices)
  + [Collaborations](https://2020.igem.org/Team%3APuiChing_Macau/Collaborations)
  + [Education](https://2020.igem.org/Team%3APuiChing_Macau/Education)
* Lab

  + [Parts](https://2020.igem.org/Team%3APuiChing_Macau/Parts)
  + [Notebook](https://2020.igem.org/Team%3APuiChing_Macau/Notebook)
  + [Safety](https://2020.igem.org/Team%3APuiChing_Macau/Safety)
  + [Protocol](https://2020.igem.org/Team%3APuiChing_Macau/Protocol)
* About Us

  + [Team](https://2020.igem.org/Team%3APuiChing_Macau/Team)
  + [Attributions](https://2020.igem.org/Team%3APuiChing_Macau/Attributions)
* [Judging Form](https://igem.org/2020_Judging_Form?id=3503)

![](https://static.igem.org/mediawiki/2020/thumb/6/63/T--PuiChing_Macau--Decor--1.png/800px-T--PuiChing_Macau--Decor--1.png)

Engineering Success

## Investigate the potential of synthesized amino acids-based flame retardants for fabrics

With the overall project goal of creating a flame retardant product that is sustainable, environmentally friendly, and efficient, we have reviewed all our possible approaches within our resource limitation. Considering polymeric fabrics material products being ubiquitous in different fields of day-to-day life, yet a majority of them carry a limitation of being flammable. In the prevention of these potential threats to user safety, essential modulation is approached, aiming to reduce polymeric material's flammability through a new manufacturing process and new multi-functional materials. However, this requires innovative molecular design, sophisticated synthesis, and therefore long hour input and expensive resources. Therefore, we carried out the investigation to:

• Validate the feasibility of amino acid based flame retardant.

• Generate basic data for future protein flame retardancy ranking system.

• Establish prototype and procedure for flame retardancy testing in our school lab.

---

**⭐ Please click the buttons below to show detailed information ⭐**

Research
Imagine & Design
Build & Test (1st Cycle)
Learn & Improve (1st Cycle)
Build & Test (2nd Cycle)
Learn & Improve (2nd Cycle)

## Potential Factors

Taking into account the potential factors causing the fire, it can be divided into “Geographical factors”, “Natural factors” and “Materials”. According to our opinions, fire retardancy will be influenced by several factors, such as the structure of the surroundings, humidity, temperature, weather, and material, etc.

### 1. Geographical factors:

Unlike humans, fires usually travel uphill much faster than downhill. The steeper the slope is, the faster the fire travels. The speed of the fire travels would double when a hill is going upwards at a 10% gradient. In the same way, if a slope is going upwards at a 20% gradient, it would quadruple the speed of the fire. It is because smoke and heat that are caused by the fire are rising uphill. As a result, the fuel further up the hill had been pre-heated before the fire approached. In addition, it is obvious that the occurrence of fire depends on the layout of the surrounding environment. For example, the closer the trees in the forest are arranged, the trees nearby are more likely to be burned when wildfires happen.

### 2. Natural factors

The higher percentage of the humidity in the air, they are higher moisture in the fuels and the less likely they are to catch fire. As a result, when it’s autumn and winter, wildfires are more likely to occur. On the other hand, the influence of season and temperature are mutually influencing. In some areas, extremely high temperatures may be reached in summer. The heat will evaporate the water in the trees, which will dry out the trees and leaves causing a fire. For example, in many parts of West Africa, the dry drought of the Sahara Desert during the dry season has begun to make fire burning easier.

### 3. Materials

One of the important factors here is the flammability of materials. The flammable items at home were mainly curtains, bed sheets, furniture, etc. With ranking of the fabrics (from easiest to hardest flammability) demonstrated: Linen →Cotton → Wool → Silk → Polyester Fabrics

Current researches established by other people all over the world have already conducted sufficient research on the natural factors affecting fire. Considering our constraints, we are prohibited from building a model to test the influence of slope or surrounding layout. Therefore, we decided to conduct burning experiments on different materials, mainly wood and polyester fabrics, which are the main material at home.

## Fire Standard & Testing Method

For testing, we employ ASTM-D6413 which is called the Standard Test Method for Flame Resistance of Bedsheets (Vertical Burning Test). After we considered our limited equipment and time and the real situation in target applicative places, we anticipate that ASTM-D6413 is the most suitable testing method for us to test our proteins/ amino acids.

The ASTM-D6413 standard is an American standard for testing the Flame Resistance of Bedsheets. In our city, we don’t have our own standard and our adjacent cities like Hong Kong, Taiwan, and so on both create their own standards that refer to the US one. The further industry application realization also provided us with the details of fire retardants’ criteria, of which at the moment they are meeting the FSD requirements from Hong Kong as Macau does not consist of a consummate institution.

As a result, we both agreed that this test is the most accessible and accurate one, which is American Standards ASTM-D6413.

According to the ASTM-D6413 authoritative documents, it is mentioned that:

1 This test method determines the response of bedsheets to a standard ignition source, deriving measurement values for after flame time, afterglow time, and char length.

---

2 The vertical flame resistance, as determined by this test method, only relates to a specified flame exposure and application time.

---

3 This test method maintains the specimen in a static, draft-free, vertical position and does not involve movement except that resulting from the exposure.

---

4 Test Method D6413 has been adopted from the Federal Test Standard No. 191A method 5903.1, which has been used for many years in acceptance testing.

---

Conferring the descriptions of this test in the ASTM INTERNATIONAL website which is <https://www.astm.org/Standards/D6413.htm>. This information validates our use of this vertical flame resistance of the bedsheets. Our experiment’s safety and accuracy can be conferred based on the reference to this standard. Although this test does have drawbacks listed in 5.4, it is however the most feasible method that enables us to conduct it in our school laboratory.

## Imagine: Develop Possible Solutions & Design: Select a Promising Solution

![](https://static.igem.org/mediawiki/2020/thumb/7/71/T--PuiChing_Macau--wiki2.jpg/800px-T--PuiChing_Macau--wiki2.jpg.png)![](https://static.igem.org/mediawiki/2020/thumb/d/d6/T--PuiChing_Macau--wiki1.jpg/800px-T--PuiChing_Macau--wiki1.jpg.png)

Biomaterial such as protein have been reported as an alternative option for fire retardant because of the high amount of phosphorus and nitrogen, which would generate thermally stable char during combustion as a protective layer and inhibits oxygen supply.

According to Xu et al (2019), several novel Nitrogen-containing amino acids (glycine, aspartic acid, and lysine) with specifically designed group molecules have been demonstrated their potential as a flame retardant for cotton fabrics via char formation. This result showed that an amino acid-based flame retardant is feasible. Amino acids are the building block of protein, each consists of an alpha carbon atom, an amino group, a carboxyl group, a hydrogen atom, and a variable component called side chain. In proteins, multiple amino acids are linked together by biochemically induced peptide bonds to form a long chain. The linear sequence of amino acids in a protein is considered as the primary structure of the protein (Nature Education, 2014). In a review of other literature, there were suggestions that milk protein would also be an option of flame retardant. Since amino acid is the basic building block of protein, we hypothesis that amino acid-based flame retardants would be feasible material choices.

Due to the COVID-19 pandemic, we decided to use the syntheized amino acid to conduct our experiments on fabrics instead of the conventional e.coli produced material to prove our hypothesis.

## Information about the Prototype

### 1. 0.25m, 0.5m and 1m Concentration of Amino Acids will be tested

Before the experiments, we tried different concentration of amino acid and found some amino acids have lower solubility. In order to completely dissolve the amino acids and make the experiment fairer and more consistent, we decided to use concentrations of 0.25m, 0.5m and 1m.

### 2. Only 5 amino acids will be tested

For the purpose of making sure that our experiments are accurate, we burn each amino acid three times at each concentration. However, that would be a huge amount. Because of the limited amino acids, we chose five amino acids with the consideration of nitrogen percentage.

### 3. Materials-Used to make the Equipment

As mentioned, the vertical testing with ASTM-D6413 will be employed. Therefore, we referred to the required equipment and built a similar device ourselves. In order to minimize the influence of external factors on the experiment such as oxygen level, we process the experiment inside the tin box made by ourselves with only one side for ventilation. It is also convenient for us to do experiments and take photos.

Materials to make the equipment: Tin Foil, Lab Racks, Alcohol Lamp, Fire Mat

### 4. Experimental Steps (including Adjustment of the Fire Testing)

We tried to ignite the bedsheets for 12 seconds according to ASTM D6413. However, we found that the whole bedsheets were burned. It is quite difficult for us to analyze. Finally, we changed it to 5 seconds.

### The above adjustment can also be regarded as a cycle. In order to make our description clear, We summarized the above content rather than showing all the experimental results which are less valuable

## Experimental steps

1. The solution was made in a beaker and the bedsheets were soaked in the beaker for a day to ensure complete absorption.

2. The bedsheets were taken out and put on the lab racks

3. The equipment made by ourselves was set up

4. The bedsheets were hold using four clips and hole by the lab racks

5. The bedsheets were ignited for 5 seconds and put out

6. Step 4 and 5 were repeated for every soaked bedsheet

In total, there are two people to conduct the experiment. One person is responsible for lighting the alcohol lamp and removing the alcohol lamp after burning for 5 seconds. The other one is responsible for video recording and timing.

![](https://static.igem.org/mediawiki/2020/thumb/5/57/T--PuiChing_Macau--BuildnTest--1.jpg/800px-T--PuiChing_Macau--BuildnTest--1.jpg)
![](https://static.igem.org/mediawiki/2020/thumb/7/76/T--PuiChing_Macau--BuildnTest--2.jpg/800px-T--PuiChing_Macau--BuildnTest--2.jpg)
![](https://static.igem.org/mediawiki/2020/thumb/6/6e/T--PuiChing_Macau--BuildnTest--3.jpg/800px-T--PuiChing_Macau--BuildnTest--3.jpg)
![](https://static.igem.org/mediawiki/2020/thumb/f/f5/T--PuiChing_Macau--BuildnTest--5.jpg/800px-T--PuiChing_Macau--BuildnTest--5.jpg)
![](https://static.igem.org/mediawiki/2020/thumb/5/58/T--PuiChing_Macau--BuildnTest--4.jpg/450px-T--PuiChing_Macau--BuildnTest--4.jpg)
![](https://static.igem.org/mediawiki/2020/thumb/6/67/T--PuiChing_Macau--chamber_photo.jpg/333px-T--PuiChing_Macau--chamber_photo.jpg.png)

## Learn & Analysis

Placed the bedsheets in trays and soaked the bedsheets with different amino acids overnight to let them coated with the amino acid. According to the IR results of different 1M amino acid, the nitrogen percentage of Arginine is the highest, thus the bedsheets coated with it took the longest time to be fully burnt out.

![](https://static.igem.org/mediawiki/2020/thumb/b/b1/T--PuiChing_Macau--amino_IR.jpg/800px-T--PuiChing_Macau--amino_IR.jpg.png)
IR of Bedsheets soaked with different Amino Acids

Generally, the average burning time increases as the nitrogen percentage increases. The most significant increase trend in which the concentration is 1m, followed by 0.5m and 0.25m. In terms of R-value, 1m concentration has the highest value which is 0.788, which means that the relationship between burning time and nitrogen percentage is mostly related during the burning testing for 1m concentration. Although the r value for the other two is low, this does not indicate that the relationship is less. Theoretically, they must be related. It can only be commented that the influenced caused by human error and unprofessional equipment have a more significant impact on the burning test for lower concentration. This can also be reflected in the scattered data, especially the 0.5m and 0.25m concentration. In terms of the trend line of 1m, the burning time suddenly increases when the nitrogen level is 21.2%. For 0.5m, the burning time is keep decreasing with the increase in the nitrogen level. Until 21.2% nitrogen level, burning time starts to increase. In the aspect of 0.25m, the data points are fluctuation. In overall, it can be said that the data points are slightly in line with the theory although the data points are scattered. It is worth to mention that the burning time of the bedsheet soaked with water is only around 17second only. Therefore, all the bedsheets soaked with different amino acids have a better fire retardancy compared to the bedsheets soaked with water.

![](https://static.igem.org/mediawiki/2020/thumb/7/71/T--PuiChing_Macau--def.jpg/800px-T--PuiChing_Macau--def.jpg.png)
Correlation of Nitrogen Level and Burning Time

![](https://static.igem.org/mediawiki/2020/4/40/T--PuiChing_Macau--Analysis--4.jpg)
Burning Test Data with the Order of Nitrogen Level

Based on the concept, it is assumed that the burning time increases when the concentration of amino acid increases. As shown in the graph and table, the average burning time for 3 different concentrations with respect to 5 different amino acids is presented. Based on these data points, their R-value is also estimated. According to the data, the R-values of L-Asparagine, L-Arginine and L-Alanine are about 0.9936, 0.9878 and 0.9449 respectively, which indicate strong positive linear relationships. In contrast, L-Serine and Glycine present relatively lower R-value, which is 0.7289 and 0.5. However, they still remain strong positive linear relationships overall. In terms of L-Arginine and L-Asparagine, their burning time increases with the increase in concentration. For the other three, their burning times are not totally meet the theory because some burning times are lower/higher with the increase/decrease of concentration. As mentioned above, the burning time of the bedsheet soaked with water is around 17 second. This means the fire retardancy of all bedsheets soaked with different amino acid is better compared to the bedsheets soaked with water.

![](https://static.igem.org/mediawiki/2020/thumb/6/60/T--PuiChing_Macau--abc.jpg/800px-T--PuiChing_Macau--abc.jpg.png)
Correlation of Concentration and Burning Time

![](https://static.igem.org/mediawiki/2020/5/52/T--PuiChing_Macau--Analysis--2.jpg)
Burning Test Data with the Order of Concentration

## Improve & Suggestion

Based on the data in the first cycle, the result basically fit the theory. The higher the solution concentration or the nitrogen percentage of the amino acid, the longer the burning time. After our first cycle, the epidemic in Macau is basically under control. Therefore, we can also make the protein in the laboratory at the University of Macau. As protein consists of several amino acids, we decided to verify whether the effect of burning bedsheets soaked with protein is better. This can also make up for the lack of burning the bedsheets soaked with protein.

## Information about the Prototype

### Two Types of Protein: SR & Alpha-Caesin

### Two Types of Adhesion Domain: Mfp5 & CBD

### Control: Deionised Water and pET-11a

## Experimental steps

 **Since only the soaking solution is different, the experiment procedure is the same as the first cycle**

1. The solution was made in a beaker and the bedsheets were soaked in the beaker for a day to ensure complete absorption.

2. The bedsheets were taken out and put on the lab racks

3. The equipment made by ourselves was set up

4. The bedsheets were hold using four clips and hole by the lab racks

5. The bedsheets were ignited for 5 seconds and put out

6. Step 4 and 5 were repeated for every soaked bedsheet

In total, there are two people to conduct the experiment. One person is responsible for lighting the alcohol lamp and removing the alcohol lamp after burning for 5 seconds. The other one is responsible for video recording and timing.

![](https://static.igem.org/mediawiki/2020/thumb/5/57/T--PuiChing_Macau--BuildnTest--1.jpg/800px-T--PuiChing_Macau--BuildnTest--1.jpg)
![](https://static.igem.org/mediawiki/2020/thumb/7/76/T--PuiChing_Macau--BuildnTest--2.jpg/800px-T--PuiChing_Macau--BuildnTest--2.jpg)
![](https://static.igem.org/mediawiki/2020/thumb/6/6e/T--PuiChing_Macau--BuildnTest--3.jpg/800px-T--PuiChing_Macau--BuildnTest--3.jpg)
![](https://static.igem.org/mediawiki/2020/thumb/f/f5/T--PuiChing_Macau--BuildnTest--5.jpg/800px-T--PuiChing_Macau--BuildnTest--5.jpg)
![](https://static.igem.org/mediawiki/2020/thumb/5/58/T--PuiChing_Macau--BuildnTest--4.jpg/450px-T--PuiChing_Macau--BuildnTest--4.jpg)
[

](https://static.igem.org/mediawiki/2020/f/f5/T--PuiChing_Macau--Fire_Test.mp4)

Left:CBD-SR;   Middle:pET11a;   Right:Deionised Water

## Learn & Analysis

According to the data, we confirmed the fire retardancy can be improved with our fire retardance protein, the material with fire retardance protein have a great improvement on fire-resisting. Furthermore, we found out that with protein with adhesion domain can last longer time, especially in CBD- SR,CBD Alpha casein have a great deal of fire defending, following with mfp5-SR, which can prove that adhesion domain can help with the fire retardant. . The detailed analysis can be referred to the [Result](https://2020.igem.org/Team%3APuiChing_Macau/Results) Part.

## Improve & Suggestion

Based on the analysis of the first and second cycle mentioned, our data are slightly in line with the theory. In order to increase the accuracy of the burning test, professional equipment is required. For this time, it is impossible for us to control the oxygen level, temperature and humidity during the experiment. In addition, the moisture content of the texture is also expected to be the same. In the aspect of data recording, the burning times is recorded artificially. If it is feasible, a flame sensor, such as One Way DC 12V Flame Sensor Relay Module or 5 Way Flame Sensor Module as shown below, can also be applied for the purpose of recording the burning time accurately

![](https://static.igem.org/mediawiki/2020/thumb/6/66/T--PuiChing_Macau--Improve--1.jpg/240px-T--PuiChing_Macau--Improve--1.jpg)![](https://static.igem.org/mediawiki/2020/thumb/8/86/T--PuiChing_Macau--Improve--2.jpg/240px-T--PuiChing_Macau--Improve--2.jpg.png)

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* Project
  + [Description](https://2020.igem.org/Team%3APuiChing_Macau/Description)
  + [Design](https://2020.igem.org/Team%3APuiChing_Macau/Design)
  + [Model](https://2020.igem.org/Team%3APuiChing_Macau/Model)
  + [Eng. Success](https://2020.igem.org/Team%3APuiChing_Macau/Engineering)
  + [Implementation](https://2020.igem.org/Team%3APuiChing_Macau/Implementation)
  + [Rslt. & Contrib.](https://2020.igem.org/Team%3APuiChing_Macau/Contribution)
  + [Prf. of Concept](https://2020.igem.org/Team%3APuiChing_Macau/Proof_Of_Concept)
* Outreach
  + [Human Practices & IHP](https://2020.igem.org/Team%3APuiChing_Macau/Human_Practices)
  + [Collaborations](https://2020.igem.org/Team%3APuiChing_Macau/Collaborations)
  + [Education](https://2020.igem.org/Team%3APuiChing_Macau/Education)
* Lab
  + [Parts](https://2020.igem.org/Team%3APuiChing_Macau/Parts)
  + [Notebook](https://2020.igem.org/Team%3APuiChing_Macau/Notebook)
  + [Safety](https://2020.igem.org/Team%3APuiChing_Macau/Safety)
  + [Protocol](https://2020.igem.org/Team%3APuiChing_Macau/Protocol)
* About Us
  + [Team](https://2020.igem.org/Team%3APuiChing_Macau/Team)
  + [Attributions](https://2020.igem.org/Team%3APuiChing_Macau/Attributions)
* [Judging Form](https://igem.org/2020_Judging_Form?id=3503)

* @iGEMPuiChing2020

* igem.wmc@g.puiching.edu.mo

© 2020 iGEM PuiChing Macau Media Team

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:PuiChing Macau/Experiments

# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

### What should this page contain?

* Protocols
* Experiments

### Inspiration

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:PuiChing Macau/Contribution

[![iGEM PuiChing Macau](https://static.igem.org/mediawiki/2020/d/d2/T--Puiching_Macau--logo.png)](https://2020.igem.org/Team%3APuiChing_Macau)

* Project

  + [Description](https://2020.igem.org/Team%3APuiChing_Macau/Description)
  + [Design](https://2020.igem.org/Team%3APuiChing_Macau/Design)
  + [Engineering Success](https://2020.igem.org/Team%3APuiChing_Macau/Engineering)
  + [Results & Contributions](https://2020.igem.org/Team%3APuiChing_Macau/Contribution)
  + [Model](https://2020.igem.org/Team%3APuiChing_Macau/Model)
  + [Implementation](https://2020.igem.org/Team%3APuiChing_Macau/Implementation)
  + [Proof of Concept](https://2020.igem.org/Team%3APuiChing_Macau/Proof_Of_Concept)
* Outreach

  + [Human Practices & Integrated HP](https://2020.igem.org/Team%3APuiChing_Macau/Human_Practices)
  + [Collaborations](https://2020.igem.org/Team%3APuiChing_Macau/Collaborations)
  + [Education](https://2020.igem.org/Team%3APuiChing_Macau/Education)
* Lab

  + [Parts](https://2020.igem.org/Team%3APuiChing_Macau/Parts)
  + [Notebook](https://2020.igem.org/Team%3APuiChing_Macau/Notebook)
  + [Safety](https://2020.igem.org/Team%3APuiChing_Macau/Safety)
  + [Protocol](https://2020.igem.org/Team%3APuiChing_Macau/Protocol)
* About Us

  + [Team](https://2020.igem.org/Team%3APuiChing_Macau/Team)
  + [Attributions](https://2020.igem.org/Team%3APuiChing_Macau/Attributions)
* [Judging Form](https://igem.org/2020_Judging_Form?id=3503)

![](https://static.igem.org/mediawiki/2020/thumb/6/63/T--PuiChing_Macau--Decor--1.png/800px-T--PuiChing_Macau--Decor--1.png)

Results and Contributions

## Overview

This study describes the construction and characterization of several new BioBricks with adhesion domain, verifying the adhesion with RFP after washing or soaking treatment. We also further performed fire retardancy tests using our engineered fire retardancy proteins, including a vertical burning test with bed sheets and a wood burning test collaborating with professional quality checking organization. Our findings here suggest that our new Bio-Bricks were successfully expressed and were functional, as shown by the results below.

## Background

Based on the achievement of iGEM 2015 Mingdao, a basic flame retardant bacterial system (using SR protein; [BBa\_K1608000](http://parts.igem.org/Part%3ABBa_K1608000) and [BBa\_K1608002](http://parts.igem.org/Part%3ABBa_K1608002)) was proposed and we are here to further improve it. We also used the Biobricks (alpha s1 casein protein; [BBa\_K2924026](http://parts.igem.org/Part%3ABBa_K2924026)) from Duesseldorf iGEM 2019 's synthetic cheese and milk projects to develop our flame retardant E. coli, as previous papers[1] suggest the casein protein to be potential green, harmless flame retardants. However, the major concern about using these fire-retardant proteins is that proteins can be easily can hardly maintain on objects and can be easily washed away. To improve the long-term fire retardancy of our protein, we added an adhesion domains to our target proteins (expected to work as predicted by our [modelling results](https://2020.igem.org/Team%3APuiChing_Macau/Model)). As one of the adhesion domain, we here used Mussel adhesive protein(mfp-5), which contains a large amount of catecholic amino acid, Dopa, in their protein sequences. Catechol offers consistent adhesion to various substrate surfaces[2]. We also used the cellulose-binding domain, a part of the CBM3 family, is a widely used protein for water contaminants removal by fusion with cell adhesion peptides, enzymes, and antimicrobial peptides[3].

## Aim

In this project, our aim is to develop an engineered recombinant E. coli strain that allows the production of flame retardant protein, capable of adhering on different surfaces. To ensure satisfactory fire retardancy in different aspects, the protein must be able to persist on different surfaces for a prolonged period of time. We, therefore, fused the flame retardant proteins with strong adhesive proteins, such as mussel adhesive protein ([BBa\_K197018](http://parts.igem.org/Part%3ABBa_K197018)) and cellulose-binding domain ([BBa\_K1478001](http://parts.igem.org/Part%3ABBa_K1478001)). To do this, we transformed flame retardant protein, together with surface adhesion protein (cellulose-binding domain and mfp5), into E. coli to enable the E. coli to produce flame retardant protein CBD-SR([BBa\_K3503004](http://parts.igem.org/Part%3ABBa_K3503004)), mfp5-SR([BBa\_K3503006](http://parts.igem.org/Part%3ABBa_K3503006)), mfp5-Alpha casein([BBa\_K3503007](http://parts.igem.org/Part%3ABBa_K3503007)), and CBD-Alpha casein([BBa\_K3503008](http://parts.igem.org/Part%3ABBa_K3503008)). In addition, to test the adhesion properties of these engineered proteins, we also added three parts with RFPs: alpha casein-RFP([BBa\_K3503009](http://parts.igem.org/Part%3ABBa_K3503009)), CBD-alpha casein-RFP ([BBa\_K3503010](http://parts.igem.org/Part%3ABBa_K3503010)) and mfp5-alpha casein-RFP ([BBa\_K3503011](http://parts.igem.org/Part%3ABBa_K3503011))

## Results

### Development of different flame retardant protein-producing E. coli strains

BioBrick information from the iGEM registry was used as reference materials to design our novel engineered flame retardant proteins. Based on our selection, SR protein from the 2015 Mingdao iGEM team and the casein protein from the 2019 Duesseldorf iGEM team were chosen as the core building blocks of our protein.

DNAs of all novel BioBricks were ordered from Integrated DNA Technologies (IDT, State of Lowa) using the iGEM sponsor credit (except BBa\_K3503009, BBa\_K3503010, BBa\_K3503011 were ordered from genscript). The DNA fragments with ampicillin-resistant protein were then cloned into the pet11a vector, followed by transformation into E.coli strain BL21(DE3) and an antibiotic selection (Figure 1).

![](https://static.igem.org/mediawiki/2020/b/b2/T--PuiChing_Macau--Alpha_casein_ptac-SR-GST_algar_plate.jpg)

Figure 1a: LB agar plate with Ampicillin 50μg/mL (E. coli Bl21(DE3) strain with Alpha casein and Ptac-SR-GST

![](https://static.igem.org/mediawiki/2020/2/2f/T--PuiChing_Macau--mfp5-Alpha_casein_CBD-Alpha_algar_plate.jpg)

Figure 1b: LB agar plate with Ampicillin 50μg/mL (E. coli Bl21(DE3) strain with mfp5-Alpha casein and CBD-Alpha

![](https://static.igem.org/mediawiki/2020/a/ae/T--PuiChing_Macau--mfp5-SR_CBD-SR_algar_plate.jpg)

Figure 1c: LB agar plate with Ampicillin 50μg/mL (E. coli Bl21(DE3) strain with mfp5-SR and CBD-SR

### Confirmation of our designed BioBrick parts

Before moving on to protein expression, the plasmid was confirmed through the below procedures.

1. Conduct overnight culture of a single positive colony from each of our recombinant E.coli strains.
2. Extract DNA from the solution.
3. Perform enzyme digestion (mlui to nhei) with subsequent examination on the size of the inserts and their corresponding plasmid. Images of the gels are shown below (Figure 2). Two bands at around 5700 bp were found in lane 2 and 3, matching the expected size of the empty backbone at 5677 bp, while two bands at about 1.5kb in lane 2 and 3 were believed to be the inserted fragment BBa\_K3503006(mfp5-SR) at 1638 bp, and mfp5-alpha casein at 1548 bp, respectively.

![](https://static.igem.org/mediawiki/2020/f/f2/T--Puiching_Macau--gel.png)

Figure 2: Agarose gel electrophoresis (1% agarose) of double restriction enzyme-digested BioBricks
Lane 1: Invitrogen trackit 1Kb plus DNA Ladder
Lane 2: pET-11a-mfp5-SR(BBa\_K3503006)
Lane 3: pET-11a-mfp5 alpha casein (BBa\_K3503007)

Sequence construct for confirming its DNA information. As shown in Figure 3 A) and B), the sequencing result of BBa\_K3503001(Alpha Casein) and BBa\_K3503004(CBD-SR) shows that we have successfully assembled our target genes into the pET-11a vector.

![](https://static.igem.org/mediawiki/2020/5/50/T--Puiching_Macau--K3503001.png)

3a) BBa\_K3503001 (Alpha Casesin)

![](https://static.igem.org/mediawiki/2020/1/1c/T--Puiching_Macau--CBD_sequence.png)

3b) BBa\_K3503004(CBD-SR)

Figure 3: Representative sequencing results.

Due to limited time for experiment, we have only validated the BBa\_K3503006(mfp5-SR), mfp5-alpha casein, BBa\_K3503004(CBD-SR), and BBa\_K3503001(Alpha Casein) plasmid; while the plasmid with RFP (BBa\_K3503009, BBa\_K3503010, BBa\_K3503011) were sequenced by genscript.

### Validation of our expressed flame retardant protein

To validate the expressed protein of our recombinant E. coli strains, we performed SDS-PAGE (coomassie blue staining) and Western blot analysis.

Prior to performing the experiment, we cultured the recombinant E. coli with 1.0mM and 0.1mM IPTG induction for the demonstration of the result. A solution at 30°C was used to trigger the expression of K1608000(SR protein) and BBa\_K3503001(Alpha casein) until reaching O.D. 0.4 (wavelength: 600nm). The cultured samples were then lysed according to our extraction protocol, and subsequently collected its supernatant for further analysis.

![](https://static.igem.org/mediawiki/2020/9/9b/T--Puiching_Macau--SR_Blue.png)

4a) Coomassie blue staining

![](https://static.igem.org/mediawiki/2020/8/85/T--Puiching_Macau--SR_western.png)

4b) Western blot

![](https://static.igem.org/mediawiki/2020/2/2f/T--Puiching_Macau--CBD_alpha_western.png)
4c) Western blot result of CBD-SR-His(BBa\_K3503004)

Figure 4: Results of inducible expression of SR containing proteins validation. A)-B) Lane 1: Bio rad protein dual color ladder, Lane 2: pet11a, Lanes 3-4: SR(K1608000), Lanes 5-6: mfp5-SR(BBa\_K3503006), Lanes 7-8: CBD-SR(BBa\_K3503004), (Lane 4,6,8 are with 1mM IPTG induction.)

As shown in Figure 4a, the result of coomassie blue shows a slight difference among the 8 bands. We performed Western Blot to confirm the result. To facilitate the detection process, a “His tag” that allowed targeting by a commercially available antibody was added to our constructs, which also enabled us to conduct western blot (Fig 4b). At 53.82 kD, we have identified a band in lane 3 and 4 which corresponded to the approximate expected size of K1608000. We had also noticed a band at 60.13 kD in lanes 5 and 6, which was the expected size of mfp5-SR(BBa\_K3503006). Finally, a band at 53.74 kD in lanes 7 and 8 were observed, showing approximately the same size as expected for CBD-SR(BBa\_K3503004).

![](https://static.igem.org/mediawiki/2020/d/d5/T--Puiching_Macau--alpha_blue.png)

5a) Coomassie blue staining

![](https://static.igem.org/mediawiki/2020/9/9d/T--Puiching_Macau--Alpha_western.png)

5b) Western blot

Figure 5: Results of inducible expression of Alpha Casein containing proteins validation.Lane 1: Bio rad protein dual color ladder, Lane 2: pet11a, Lanes 3-4: Alpha casein(BBa\_K3503001), Lanes 5-6: mfp5-Alpha casein(BBa\_K3503007), Lanes 7-8: CBD-Alpha casein(BBa\_K3507008), (Lane 4,6,8 are with 0.1mM IPTG induction.)

As shown in Figure 5a, the result of coomassie blue also showed a slight difference among the 8 bands. We performed Western Blot to confirm the result. At 25.42 kD, we found a band in lane 3 and 4 which corresponded to the approximate expected size of Alpha Casein(BBa\_K3503001)(Fig 5b). Two bands at 60.13 kD were found in lanes 5 and 6, which was the expected size of mfp5-Alpha casein(BBa\_K3503007). Finally, a band at 50.58kD in lanes 7 and 8 was identified, showing approximately the same size as expected for CBD-Alpha casein(BBa\_K3507008).

![](https://static.igem.org/mediawiki/2020/0/00/T--Puiching_Macau--RFP_blue.png)
Figure 6: Results of inducible expression of RFP-Alpha Casein containing protein validation.
Lane 1: Bio rad protein dual color ladder
Lane 2: pet11a
Lanes 3-4: Alpha casein-RFP(BBa\_K3503009)
Lanes 5-6: mfp5-Alpha casein-RFP(BBa\_K3503011)
Lanes 7-8: CBD-Alpha casein- RFP(BBa\_K3507010)
(Lane 4,6,8 are with 0.1mM IPTG induction.)

To validate the Alpha casein containing proteins (Figure 6), we had performed coomassie blue. At 51.29 kD, we found two bands in lane 3 and 4 which corresponded to the approximate expected size of Alpha casein-RFP(BBa\_K3503009). Later on, we found a band at 83.16 kD in lanes 5 and 6, which was the expected size of mfp5-Alpha casein-RFP(BBa\_K3503011). Finally, a band at 76.76 kD in lane 7 and 8 was identified, showing approximately the same size as expected for CBD-Alpha casein- RFP(BBa\_K3507010).

### Investigation of the flame retardancy of our engineered protein

To investigate the flame retardancy of our engineered protein, IPTG was added to the bacterial cultures. IPTG addition was achieved at a time of 6 hours to reach the concentration of 1mM and incubate overnight. The culture samples were then lysed according to our extraction protocol and collected its supernatant and pellet.

As our project was continuously shaped by "[Human Practices](https://2020.igem.org/Team%3APuiChing_Macau/Human_Practices)", our team has then focused on selecting fabric materials, with a final choice “bed sheet” as the subject to test the flame retardancy of our engineered proteins. In addition, our team has developed a testing prototype and protocol through the achievement of "[Engineering Success](https://2020.igem.org/Team%3APuiChing_Macau/Engineering)" and applied this method to conduct our experiment.

Briefly, we placed the bed sheets in trays and soaked the bed sheets with different proteins and let dried overnight (Figure 7a and 7b). We then compared the IR spectrum between water and protein-coated bed sheets. As shown in the figure 7c, all bed sheets soaked in proteins solution showed characteristic of protein (smaller wavernumber at around 3300 and 1400-1600 when comparing with the water control) after dried, indicating a successfully of coating. Interestingly, we also found the wavenumber of SR protein series (SR protein, mfp5-SR, CBD-SR) has a larger changes on the absorbability of the energy than the alpha casein series (alpha casein, mfp5-alpha casein, CBD-alpha casein), which means that the SR protein contains higher number of Nh bond and thereby may indicating a higher nitrogen level in general, consistent with what we found when [designing](https://2020.igem.org/Team%3APuiChing_Macau/Design) our project.

![](https://static.igem.org/mediawiki/2020/thumb/b/bd/T--PuiChing_Macau--dry.jpg/800px-T--PuiChing_Macau--dry.jpg)

Figure 7a.Placed the bed sheets in trays and soaked the bed sheets with different proteins

![](https://static.igem.org/mediawiki/2020/thumb/8/81/T--PuiChing_Macau--dry1.jpg/799px-T--PuiChing_Macau--dry1.jpg)

Figure 7b.Let the bed sheets dry overnight

![](https://static.igem.org/mediawiki/2020/d/da/T--PuiChing_Macau--protein_ir.jpg)
Figure 7c.IR spectrum of target engineered flame retardant on bed sheet

The bed sheet (Figure 8 and video 1) being tested was in the size of 300mm\*130mm with moisture content percentage 13.55-14.37%. As demonstrated in Fig. 8, all bed sheets coated with our engineered flame retardant proteins (BBa\_K3503001 Alpha-s1-casein-His, BBa\_K3503004 CBD-SR-His, BBa\_K3503006 mfp5-SR-His, BBa\_K3503007 mfp5-alpha-His, BBa\_K3503008 CBD-alpha casein-His) showed an improved flame retardancy compared with the water control.

![](https://static.igem.org/mediawiki/2020/e/eb/T--PuiChing_Macau--Poster_Fire11.png)
Figure 8: Vertical burning test using bed sheet (BBa\_K3503001 Alpha-s1-casein-His, BBa\_K3503004 CBD-SR-His, BBa\_K3503006 mfp5-SR-His, BBa\_K3503007 mfp5-alpha-His, BBa\_K3503008 CBD-alpha casein-His )
[

](https://static.igem.org/mediawiki/2020/f/f5/T--PuiChing_Macau--Fire_Test.mp4)

Video 1. Representative videos of our vertical burning test. Left:CBD-SR-His (K3503006);   Middle:pET11a;   Right:Deionised Water

Wooden furniture is commonly used in households, offices, and the hotel industry. In addition, wood is a common building material in some countries. Therefore, we have also tested our engineered proteins on wood, in collaboration with professional quality checking organization (IDQ) in Macau SAR. The test being used here is the Bs476-part4:1970 test, a test commonly used as a fire retardancy test for building materials and structures (non-combustibility test for materials). In general, a piece of wood under 450 degrees of heat gets burned in about 5 minutes. In our fire retardant test for wood coated with different proteins, these wood pieces got burned in more than 8 minutes. This test demonstrated the fire retardancy of our proteins. The test object was a wooden block with a size of 40mm\*38mm\*51mm and the average moisture content percentage was 12.6%. Due to the limitation of our resources and time, we only prepared enough samples for Alpha casein, Mfp5-Alpha casein, and CBD-Alpha casein. As demonstrated in Fig. 9, all wood coated with our engineered flame retardant proteins displayed an improvement in flame retardancy compared with the water control.

![](https://static.igem.org/mediawiki/2020/a/af/T--PuiChing_Macau--Poster_Fire3.png)
Figure 9. Fire retardant test for wood

![](https://static.igem.org/mediawiki/2020/4/46/T--Puiching_Macau--wood_test1.png)

Before test

![](https://static.igem.org/mediawiki/2020/8/8d/T--Puiching_Macau--wood_test2.png)

The wood smoldered

![](https://static.igem.org/mediawiki/2020/thumb/7/72/T--Puiching_Macau--test.png/1600px-T--Puiching_Macau--test.png.jpeg)

The wood burned

### Investigation of the improvement of flame retardancy with additional surface adhesive function

In order to find out the adhesion properties of the engineered proteins, we ran an adhesion test, using Nikon A1MP + fluorescence confocal microscope (Nikon, Japan). We tested fabric material (bed sheets) washed and soaked. We choose to use a red fluorescent protein for checking because the material we used for the test has very few red fluorescents before treatment. Therefore, we can have a great comparison for the test.

As shown in Figure 10, the protein has successfully attached to the bed sheet. We can see that there are differences after washing and soaking, the adhesion had both become weaker after the process in 92.9% after soaking and 56.7% after washing for mfp5-Alpha casein-RFP(BBa\_K3503011) while the intensity of CBD-Alpha casein-RFP(BBa\_K3503010) after soaking and washing was 80.9% and 59.9%, respectively. Compared with control, the intensity declined dramatically after the process, to only 12.7% and 11.7%.
As we can see in figure 10, these are the results observed by the fluorescence microscope after washing, it is clear that the adhesion domain added alpha casein remains a great amount of protein on the fabrics. On the other hand, the alpha casein that without adhesion domain were washed away.

Overall, the successful attachment of the adhesion domain to the material and great improvement in adhesion was demonstrated.

![](https://static.igem.org/mediawiki/2020/6/66/T--Puiching_Macau--The_percentage.png)

10a)The percentage of the protein after processes

![](https://static.igem.org/mediawiki/2020/7/7a/T--Puiching_Macau--alpha_RFP.png)

10b)RFP-Alpha Casein(BBa\_K3503009) by different state of process(non- washing, after washing with water and soaking.)

![](https://static.igem.org/mediawiki/2020/0/04/T--Puiching_Macau--mfp5_RFP.png)

10c)RFP-mfp5-Alpha casein(BBa\_K3503011) by different state of process(non- washing, after washing with water and soaking.)

![](https://static.igem.org/mediawiki/2020/f/f1/T--Puiching_Macau--CBD_RFP.png)

10d)BBa\_K3503010 (RFP-CBD-Alpha casein) by different state of process(non- washing, after washing with water and soaking.)

Figure 10. Adhesion of the protein after processing.

![](https://static.igem.org/mediawiki/2020/6/63/T--Puiching_Macau--Alpha_RFP.png)

(a)Alpha Casein-RFP(570-620nm)

![](https://static.igem.org/mediawiki/2020/c/c0/T--Puiching_Macau--alpha_RFP_bright.png)

(b)Alpha Casein-RFP(bright field)

![](https://static.igem.org/mediawiki/2020/e/ea/T--Puiching_Macau--mfp5_red.png)

(a)mfp5-Alpha casein-RFP(570-620nm)

![](https://static.igem.org/mediawiki/2020/0/0b/T--Puiching_Macau--mfp5_bright.png)

(b)mfp5-Alpha casein-RFP(bright field)

![](https://static.igem.org/mediawiki/2020/3/30/T--Puiching_Macau--CBD_red.png)

(a)CBD-Alpha casein-RFP(570-620nm)

![](https://static.igem.org/mediawiki/2020/0/02/T--Puiching_Macau--CBD_bright.png)

(b)CBD-Alpha casein-RFP(bright field)

Figure 11. The red fluorescence in 570nm-620nm

## Conclusion

Overall, we have confirmed the successful expression of our engineered flame retardant proteins from the designed recombinant E.coli strains. These engineered proteins have demonstrated an observable improvement on fire-resisting for bed sheet and wood, compared to control. Moreover, we found out that the addition of the cellulose-binding domain to our flame retardant would improve the adhesive level of our flame retardant proteins to the fabrics and therefore improved the lifespan of our flame retardant proteins.

## References

[1] Basak, S., & Ali, S. W. (2016). Sustainable fire retardancy of textiles using bio-macromolecules. Polymer Degradation and Stability, 133, 47-64.
[2] Lee, H., Scherer, N. F., & Messersmith, P. B. (2006). Single-molecule mechanics of mussel adhesion. Proceedings of the National Academy of Sciences, 103(35), 12999-13003.
[3] Tormo, J., Lamed, R., Chirino, A. J., Morag, E., Bayer, E. A., Shoham, Y., & Steitz, T. A. (1996). Crystal structure of a bacterial family‐III cellulose‐binding domain: a general mechanism for attachment to cellulose. The EMBO journal, 15(21), 5739-5751.

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