Team:PuiChing Macau/Poster

Poster: PuiChing_Macau

Engineering E. coli to produce safe and eco-friendly flame retardant proteins

Presented by Team PuiChing_Macau 2020

Abstract

Current flame retardant materials are known to be hazardous to human body and our environment. Other than this, fire retardant protein nowadays cannot retain on the surfaces of objects and can be washed away easily, precluding the application of these proteins. To solve this problem, we here engineered Eco-friendly and harmless flame retardant proteins (SR or alpha-casein) fused with surface adhering proteins (cellulose binding domain or mussel adhesive proteins). Thereby, we improved previous iGEM flame retardant systems, which contain only flame retardant proteins. We here proved the protein expression, fire retardancy and sustainability of our flame retardant systems, matching our previous modeling results. Additionally, we also engineered an easy-to-make vertical burning test, helping us and future iGEM teams to test flame retardants.

Team Members and their Attribution

Weng I (Alisa) LEONG : Team leader, Outreach, Result Analysis, Modeling, Filming and Wiki Page
Ieng Chon (Chloe) LI : Labwork Leader, Outreach, Result Analysis and Lab Log
Yi Fan (Linda) XIANG : Outreach Leader, Modeling
Sin Mei (Jasmine) CHEONG : Outreach and Marketing
Cho Cheng (Elly) SHE : Lab Work
Weng Seong (Yoyo) LEI : Modeling, Wiki Page and Engineering Success
Pak Chong (Jimmy) CHEONG : Outreach and Treasury
Chan In (Merry) NG : Engineering Success Leader and Modeling
Nga Chi (Angie) LEONG : Modeling, Mascot Design and Filming
Teng Wai (Corinnie) HOI : Mascot Design and Animation
Weng Si (Victoria) CHIO : Filming and Editing of Video and Engineering Success
Lok Hang CHIU : Logo Design, Filming and Editing of Video
Hou (Lucas) IONG : Outreach
Weng In LAI : Outreach
Jeremy HU : Human Practice

Introduction & Inspiration

What are fire retardants?

Fire retardants are substances designed to prolong escape time during a fire accident. In general, the fire retardants enable the prevention of fire spreading when combined with certain combustible and flammable materials, including bed sheets, furniture, electronic devices, and building materials.

However, current fire retardants are imperfect, comprised of hazardous chemicals that may impair our health and the environment. To elaborate, chemicals antimony trioxide, boric acid, zinc borate, decabromodiphenyl oxide and melamine found in inorganic fire retardants may induce cancer, neurodegenerative diseases, or organ failure. In addition, TBBPA, PBDEs, PCBs from organohalogen-based fire retardants have also been proved to be endocrine-disrupting chemicals that act as either antagonists or agonists at receptor sites of androgen, progesterone, and estrogen. In addition to this, current fire retardant cannot adhere to the surfaces of objects over time and can be washed away easily,which lower its fire retardancy. Inspired by this issue, the objective of our project is to develop an engineered recombinant E.coli strain that allows the production of a novel eco-friendly and adhesive flame retardant.

Solution

To address this problem, we engineered E. coli to produce flame retardant proteins with human SR protein and alpha-casein protein, of which, are molecules registered by the previous 2015 Mingdo iGEM team and the 2019 Duesseldorf's iGEM team, respectively. In addition to our attempt, an improvement approach of iGEM flame retardant systems was included, involving the fusion of surface adhering proteins derived from cellulose-binding domain and mussel adhesive proteins that aim for adaptation to achieve long-term retention of the protein on the object's surface. As a result, a sustainable and environmentally friendly flame retardant is developed in this project, displaying a promising future to replace traditional flame retardants and eventually improve the fire retardancy of current building materials.

Mechanism

Fire retardants are most commonly divided into three groups based on their properties and chemical structure, namely Organohalogen Flame retardants, Nitrogen flame retardants, and Phosphorus. Due to their different chemical properties, their flame retardancy mechanism is also diversified (Endothermic degradation, the establishment of thermal insulation barrier, Dilution of the gas phase, Gas-phase radical quenching).

Mechanism of nitrogen-based fire retardant and phosphorus-based fire retardant

Our project mainly focus on fire retardant which is nitrogen-based and phosphorus-based due to the toxicity and environmental factors

Nitrogen-based fire retardant

Nitrogen-containing flame retardants may act by the release of inert gases (ammonia, nitrogen) into the gas phase. The formation of these gases and the decomposition of the flame retardant absorb most of the heat, which greatly reduces the surface temperature of the polymer. Moreover, the nitrogen gas released insulate the oxygen, therefore, damage the fire triangle.

Phosphorus-based fire retardant

The high temperatures of the flame result in the chemical bonds of phosphorus-based fire retardant releasing phosphoric acid, which helps in speeding up the breakdown of cellulose and polyester. When the cellulose and polyester are broken down, it favours the production of char, which is stable at these high temperatures and form a carbonated barrier that prevents the remains of the fabric from burning.

Engineering Success

With the overall project goal of creating an environmentally friendly, sustainable and efficient flame retardant product, we have reviewed all our possible approaches within our resource limitation. Finally, we decided to conduct burning experiments on different materials to study the performance of fire retardant protein applied to different materials. Based on the limited equipment and time, we believed that ASTM-D6413 is the most suitable testing method for us to test the fire retardant performance on fabrics/bedsheets. Due to the COVID-19 pandemic, we decided to use the synthesized amino acid to conduct our experiments on bedsheets to prove our hypothesis. 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. After we conducted the burning test for the bedsheets soaked with different amino acids, we also did the same experiments for the bedsheets soaked with our designed protein because it was feasible to produce the proteins at that time.

Experimental Design

Figure 1. Vertical Burning Test

• 5 seconds ignition is adjusted instead of 12 seconds


• Before the experiments, we had tried different concentrations of amino acids. However, we found that some amino acids have lower solubility. To completely dissolve the amino acids to have a consistent experiment, we decided to use concentrations of 0.25M, 0.5M and 1M.


• Due to the limited amount of amino acids, we chose five amino acids with the consideration of nitrogen percentage, so only 5 amino acids have been tested.


• Before we started the experiment, we placed the materials into different amino acid solutions with different concentrations to let the materials fully soaked with the amino acid.


• We burn each amino acid thrice for each concentration to make our experiments more precise.

Analysis

In terms of analysis, we did two parts to estimate the fire-retardant performance of amino acids. Before the burning test, we used infrared spectroscopy (IR) to check whether amino acids stick to the bedsheets.

Figure 2. IR of Bedsheets soaked with different Amino Acids
Afterwards, the burning experiments were conducted. According to the results, the average burning time increases as the nitrogen percentage or concentration of amino acids increases generally. Comparing the concentration, the burning time of the textiles soaked with amino acids with 1m concentration had the longest burning time. Comparing different amino acids with 1m concentration, the bedsheets soaked with 1m Arginine had the best performance since its nitrogen percentage is highest. To sum up, the result showed that the higher the solution concentration or the nitrogen percentage of the amino acid is, the longer the burning time it takes.


Figure 3. Burning Test Data with the Order of Nitrogen Level

Learn & Improve

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.

Figure 4: One Way DC 12V Flame Sensor Relay Module

Figure 5: 5 Way Flame Sensor Module

Parts

Figure 1. Alpha-casein with 6*His tag part


Figure 2. Fire retardant protein with adhesion domain parts


Figure 3. Alpha-casein (with adhesion domain) and RFP parts

Modeling

We test our idea to add adhesion protein to fire retardant proteins with modeling. Thus, we here built a model to predict the efficiency of protein-based fire retardants across time to serve the following two purpose:
1. Compare and contrast the fire-retardant efficiency of various proteins.
2. By comparing proteins with and without adhesion domain, we can predict whether adhesion domains (cellulose binding domain, CBD; mussel foot protein 5, mfp5) can improve fire retardancy across time.

Assumptions

1. The experiment conditions are the same in all data used to build the model.
2. The fire retardancy decay across time is the same as the decay by washing the protein out.
3. The fire retardancy of the protein is linearly correlated with the nitrogen level of the protein.

Modeling Process

(1) Find the best equation (model) to predict fire-retardancy decay across time through the dataset from the 2019 Linkoping Sweden iGEM team (CBD-sfGFP Column Purification data). We found that the exponential decay function has the highest correlation coefficient (R). Therefore, we here used the following exponential function for our modeling:

$Adhered\; protein\; =\; alpha\; x\; ebeta\; x\; t$ (1)

(2) In order to compare proteins with and without adhesion proteins, we used the data from 2015 Imperial College London iGEM team. We fit the data with an exponential decay equation to find out all the corresponding coefficients of protein with (CBDcipA-sfGFP) and without adhesion (sfGFP) domains across time. Here are coefficients we found:

$Adhered\; protein\; with\; CBD\; =\; 0.8292\; \times \; e-0.03092t$ (2)
$Adhered\; protein\; without\; CBD\; =\; 0.9891\; \times \; e-0.7413t$ (3)

(3) Calculate fire-retardancy (F) decay across time using corresponding modes by multiplying the protein adhered(A) with the percentage of nitrogen(N), which is defined as:

$N\; =\; M$_{nitrogen in the protein}/ M_protein (4)

Therefore, the relative fire retardancy (F) would be:

$F\; =\; N\; x\; A$ (5)
$F\; =\; M$_{nitrogen in the protein}/ M_protein x alpha x e^{beta x t} (6)

Figure 1. Figure illustrating fire retardancy of protein Uncharacterized histidine-rich protein (uniprot: Q8MP30) with and without adhesion domain.

Figure 2. Figure illustrating fire retardancy of alpha-casein with and without adhesion domain.

Figure 3. Figure illustrating fire retardancy of SR protein with and without adhesion domain.
Overall, the proteins with CBD retain a higher fire retardancy than the protein without CBD across time. Therefore, we decided to add adhesion domains to the fire-retardant proteins in our wet lab project. As using different kinds of fire retardant proteins evidently does not change the fire retardancy dramatically, we decided to focus on SR protein and alpha casein, in which the fire retardancy were tested in a previous iGEM team and previous research respectively.

Functional Test

After visiting Wynn Macau, a luxury hotel and resort in Macau, we found out adhesion was also important for a successful fire retardant protein. They are spraying to update the fire retardant every two years in over $130,000 USD. Therefore, to improve the adhesiveness of the fire retardant, we have added strong adhesion protein, K1321014(cellulose binding domain)and K3089023(mussel adhesion protein) to our target protein K1608000(2015Mingdao iGEM SR protein) , K2904026 (2019 Dusseldorf iGEM Alpha casein protein)and has added 6 his tag. Figure1: Fire Retardant Treatment for Wynn Hotel
Therefore, we have added strong adhesion protein, K1321014(cellulose binding domain)and K3089023(mussel adhesion protein) to our target protein K1608000(2015Mingdao iGEM SR protein), K2904026 (2019 Dusseldorf iGEM Alpha casein protein)and has added 6 his tag.

Methodology

In order to test the fire retardancy of the protein and improved parts, we have done several fire retardant tests with fabric material (bedsheet) and wood. 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). However, the burning temperature was adjusted to 450 degree Celsius because it is difficult for us to compare the result if we use the standard one.
Figure 2: Fire retardant test of wood, collaboration with professional quality checking organization (IDQ) in Macau SAR.

Results

The bedsheet being tested was in the size of 300mm*130mm with moisture content percentage 13.55-14.37%. As demonstrated in Fig. 3, 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.
Figure 3: Vertical burning test using bedsheet (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 ) Error Bar:standard deviation sigma n=2
As demonstrated in Fig.4, all wood coated with our engineered flame retardant proteins displayed an improvement in flame retardancy compared with the water control, from 5 minutes in general to more than 8 minutes after adding our proteins. Figure 4: Vertical burning test using wood

Adhesion Test

Methodology

In order to find out the adhesion properties of the engineered proteins, we ran an adhesion test. 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. We use Nikon A1MP + fluorescence confocal microscope (Nikon, Japan) to check whether fire retardant protein (with RFP) has attached on our material and the remaining fire retardant protein (with RFP) on the material after washing or soaking.

Results

Figure 1: The percentage of the protein after processes

Figure 2: RFP-Alpha Casein(BBa_K3503009) by different state of process(non- washing, after washing with water and soaking.)

Figure 3: RFP-mfp5-Alpha casein(BBa_K3503011) by different state of process(non- washing, after washing with water and soaking.)

Figure 4: BBa_K3503010 (RFP-CBD-Alpha casein) by different state of process(non- washing, after washing with water and soaking.)

As shown in the Figures above, the protein has successfully attached to the bed sheet. It is clear that the adhesion domain added alpha casein remains a great amount of protein on the fabrics. Overall, the successful attachment of the adhesion domain to the material and great improvement in adhesion was demonstrated. As shown in figure above, the protein has successfully attached to the bed sheet. At the 570-620 nm wavelength, it indicates the intensity of fire retardant protein (with RFP). 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 K3503011(mfp5-Alpha casein-RFP) while the intensity of K3503010 (CBD-Alpha casein-RFP) after soaking and washing were 80.9% and 59.9%, respectively. Compared with control, the intensity declined significantly after the process, to only 12.7% and 11.7%.

Figure 5: CBD-Alpha casein-RFP (Before washing or soaking)
(570-620nm)

Figure 6: CBD-Alpha casein-RFP
(After washing)
(570-620nm)

Figure 7: CBD-Alpha casein-RFP
(After soaking)
(570-620nm)

As we can see in figure 5, 6, 7, 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. So we can conclude that the adhesion domain does help to the longevity of our fire retardant protein.

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.

Team Achievement

Bronze:

Competition Deliverables

• wiki
• poster
• presentation video
• project promotion video
• judging form

Attributions

https://2020.igem.org/Team:PuiChing_Macau/Attributions

Project Description:

https://2020.igem.org/Team:PuiChing_Macau/Description

Contributions:

https://2020.igem.org/Team:PuiChing_Macau/Contribution
BBa_K1608000 (SR/pSB1C3 Mingdao2015)
BBa_K1608002 (GST-SR/pSB1C3 Mingdao 2015)

Sliver

Engineering success:

1. amino acid
2. proteins & adhesion domain (water as control)

https://2020.igem.org/Team:PuiChing_Macau/Engineering BBa_K3503004 (CBD-SR-His) BBa_K3503006 (mfp5-SR-His)

Collaboration:

• HK_SSC

https://2020.igem.org/Team:PuiChing_Macau/Collaborations

Human Practices:

• Wynn Macau
• Fire Services Bureau (Government Policy)
• Institute for Development and Quality of Macau IDQ (Local Testing Centre)

https://2020.igem.org/Team:PuiChing_Macau/Human_Practices

Proposed Implementation:

https://2020.igem.org/Team:PuiChing_Macau/Implementation

Gold

Integrated Human Practices:

• Wynn Macau

https://2020.igem.org/Team:PuiChing_Macau/Human_Practices

Improvement of an Existing Part:

• Previous: BBa_K2924026 α-s1-casein (CSN1S1)
• Improved: BBa_K3503010 (CBD-alpha casein-RFP)

Project Modeling:

Relative fire retardancy of different protein with and without adhesion domain

Proof of Concept:

• vertical fire test
• woodblocks test
• modeling
• adhesive test

https://2020.igem.org/Team:PuiChing_Macau/Proof_Of_Concept

Science Communication:

• Talk at secondary school
• Booth game
• Newspaper articles
• Promotion video

https://2020.igem.org/Team:PuiChing_Macau/Education

Future Direction

1.Funding

We are currently in plan to raise funding for our project. As we hope that our product can reach the standard in Macau and provide the market a more environmentally-friendly alternative of fire retardant, most of the funding will be allocated for future research and further flame retardancy experiment. We are also planning to expand our project and profit through licensing our future product's patent.

2.Toxicity & allergy tests

In terms of the safety aspects of our project, there is still considerable room for future work as no toxicity test or allergy test have been conducted on our product due to various limitations. However, since most of the subparts involved in the protein are extracted from non-toxic precursors, we predict that our fire retardant protein is also environmentally friendly. In future, we hope to complete a standardized toxicity and allergy test eg.antigen testing of our product in order to verify its safety.

3.Extended application

Since the idea of our project was inspired by the Grenfell Tower fire in the UK 3 years ago, we are considering further applying our product into the building's exterior coating, besides usage on common household and hotel products such as fabrics material or wooden furniture. By applying our fire retardant on exterior walls of public buildings, we hope to discover some potential problems that we have not noticed before which are crucial for the future improvement of our products. Our short-term goal is to investigate the possibility to increase 'odor Eliminator' like function to our flame retardant product, e.g. protein/chemical mixture and manufacture household use flame retardant spray.

4.Future customers and collaboration

We hope to deepen our connection with various parts of society in the future. As our first few steps, we contacted the furniture company---IKEA to imply our tests by email. However, due to covid-19, we still have not received a formal replyment. Nonetheless, we have never stopped trying, we still made a visit to Macau’s fire station and collaborated with Wynn Hotel, to acknowledge more information about fire-retardants in Macau. Afterall, we understand, although our product might have a higher cost compared with other fire retardants, our product can provide a more environmentally-friendly and sustainable way to prevent fire.

5. Education

Public education about fire prevention is fundamental for ameliorating casualties and properties damage caused by fire. Anyone in the society has the duty to lend others a helping hand especially when we, as an IGEM team having the target of saving people's lives from fire, we have the responsibility to educate the public about preventing home fire and wildfire. In future, we are hoping to organize regular lectures to draw people's attention and awareness to precautions of fire.

6. Further functional tests and adjustments on fire retardant tests

Higher proficiency experiments on our fire-retardant(eg. vertical burning test) in the future are ideal and desired. Caused by the reasons for both covid-19 and the lack of standardized equipment provided, it is reasonable that our test results may have huge variation. To cope with this problem, we look forward to collaborating with more organizations and universities to have these experiments done to modify the accuracy and preciseness of our protein. Moreover, the parts of our foremost consideration, the safety to humans and the environment, should also be tested throughout these experiments and adjustments that will be carried soon.

References and Acknowledgements

Literature Sources

1. Costa, L. G., Giordano, G., Tagliaferri, S., Caglieri, A., & Mutti, A. (2008). Polybrominated diphenyl ether (PBDE) flame retardants: environmental contamination, human body burden and potential adverse health effects. Acta Biomed, 79(3), 172-183.
2. Yanosky, T. M., & Kappel, W. M. (1997). Effects of solution mining of salt on wetland hydrology as inferred from tree rings. Water Resources Research, 33(3), 457-470.
3. Sun, M., Lowry, G. V., & Gregory, K. B. (2013). Selective oxidation of bromide in wastewater brines from hydraulic fracturing. water research, 47(11), 3723-3731.
4. Ma, D., Wang, Z., Guo, M., Zhang, M., & Liu, J. (2014). Feasible conversion of solid waste bauxite tailings into highly crystalline 4A zeolite with valuable application. Waste management, 34(11), 2365-2372.
5. Carosio, F., Di Blasio, A., Cuttica, F., Alongi, J., & Malucelli, G. (2014). Flame retardancy of polyester and polyester–cotton blends treated with caseins. Industrial & Engineering Chemistry Research, 53(10), 3917-3923.
6. Basak, S., & Ali, S. W. (2016). Sustainable fire retardancy of textiles using bio-macromolecules. Polymer Degradation and Stability, 133, 47-64.
7. 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.
8. 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.

Advisors & Supports

• Primary Investigator : Wai Man Cheong
• Secondary Investigator : Seak Chi U
• Sponsor: Wynn Care
• General Support: The Science and Technology Development Fund (FDCT), Institute for the Development and Quality (IDQ), Faculty of Health Science, University of Macau, Pui Ching Middle School
• Advisors: Tsz On Lee, Ruiyu Xie, Tzu Ming Liu
• Instructor (wet lab, wiki, presentations): Lok In Lo
• Instructor (Engineering, Hardware, Matlab, Wiki): Fong Tin Cheng
• Instructor (wiki): Franklin Yeung
• Lab Support : Hao Nian Min, Weng I Lei, Hau Yin Leung, Stephanie Pei Wen Ng, Yatling Mo