Team:PuiChing Macau/Proof Of Concept
Fire retardancy
In order to verify the fire retardancy of our bio-engineered fire retardant protein, our team has focused on selecting experiment applied fabric materials. As the final choice, our team decided to test flame retardancy of our bioproduct with bedsheets. In addition, our team developed a testing prototype and protocol to further test the fire retardancy of our bioproduct. Based on referencing the current fire standardized test, we have applied “ASTM-D6413” or Standard Test Method for Flame Resistance of Textiles (Vertical Test) as our fire retardancy test. Of note, the test was selected after precise consideration of the limitations from our equipment, time and the real situation in target applicative places.
As mentioned, vertical testing with ASTM-D6413 is employed. We have referred to the required equipment and built a similar device. In construction, the device was built tin box with only one-sided ventilation, therefore minimizes the influence of external factors, such as the oxygen level (Figure 1, 2). Upon completion, the test was processed within in tin box.
Figure 1. Vertical fire test design
Figure 2. Vertical fire test setup
Figure 3. Vertical Burning Test using the bedsheet.
The test subject (Figure 3.) was the size of a bedsheet in 300*130mm and the average Moisture content% in 13.55%-14.37%. As demonstrated on Fig.3, all of our engineered flame retardants (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 ) displayed an improvement compared with the water control.
Video 1. Representative videos of our vertical burning test. Left:CBD-SR-His K3503006; Middle:pET11a; Right:Deionised Water
Figure 4. Fire retardant test for wood
We had also tested our fire retardant protein on woods (Bs476-part4:1970 test, standard non-combustibility test for materials and structures). The test object was a wooden block in size of 40*38*51mm and an average Moisture content% of 12.6%. However, due to the limited resources and time, we had only prepared enough samples for K3503001(Alpha Casein-His), K3503007(Mfp5-Alpha casein-His), and K3503008(CBD-Alpha casein). As indicated in Fig 4, all of our engineered flame retardants displayed an improvement compared with the water control.
Adhesiveness
Before we start our project, we have applied computational modeling to assess different proteins and their effectiveness in terms of their longevity, fire-retardancy and other factors. We have also tested whether it would be useful to add adhesion domains to our fire-protein in an attempt to prolong the longevity of the fire-retardant material.
Fire retardancy=Nitrogen percentage ×Adhered protein (1)Fire retardancy =(Mnitrogen in the protein/ Mprotein) x alpha × e(beta×t) (2)
Figure 5. Figure illustrating fire retardancy of alpha-casein with and without adhesion domain.
Figure 6. Figure illustrating fire retardancy of SR protein with and without adhesion domain.
As shown by the results above (Figure 5&6), the proteins with CBD has demonstrated a stable fire retardancy across time. We, therefore, believe that the genetic fusion of fire-retardant protein and adhesion domain would generate more stable fire retardant proteins. Since 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 was tested in a previous iGEM team and previous research respectively.
We had run the adhesion test with materials that had been washed and soaked, in order to find out whether our proteins have successfully fused with the adhesion domain or not. Since the bedsheet sample, we have included a limited amount of red fluorescents, hence, we thought to apply red fluorescent proteins for checking the proteins that remained on the sample after soaking or washing, aiming to enable a comparison for the test. As shown in Figure 7 that the two binding domains: CBD and mfp5 would improve the attachment of our caesin flame retardant protein's attachment to the bed sheet.
Figure 7.The percentage of the protein on bed sheet after processes
As shown in the result through Nikon A1MP+ fluorescence confocal microscope, red fluorescents remained on the sample after washing or soaking, which means that our proteins have successfully adhered to the sample and remain across times of wash.
(a)Alpha Casein-RFP(570-620nm)
(b)Alpha Casein-RFP(bright field)
(a)mfp5-Alpha casein-RFP(570-620nm)
(b)mfp5-Alpha casein-RFP(bright field)
(a)CBD-Alpha casein-RFP(570-620nm)
(b)CBD-Alpha casein-RFP(bright field)
Conclusion
Overall, we have proofed the fire retardancy of our novel fire retardant protein by the means of a vertical fire test built for ourselves and the woodblocks test performed by IDQ. Both results show an improvement in fire retardancy. Through the modeling and adhesive test, it is clear that the protein with the adhesion domain can remain on the material for a longer time.