Team:PuiChing Macau/Contribution
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 and BBa_K1608002) was proposed and we are here to further improve it. We also used the Biobricks (alpha s1 casein protein; BBa_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). 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) and cellulose-binding domain (BBa_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), mfp5-SR(BBa_K3503006), mfp5-Alpha casein(BBa_K3503007), and CBD-Alpha casein(BBa_K3503008). In addition, to test the adhesion properties of these engineered proteins, we also added three parts with RFPs: alpha casein-RFP(BBa_K3503009), CBD-alpha casein-RFP (BBa_K3503010) and mfp5-alpha casein-RFP (BBa_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).
Figure 1a: LB agar plate with Ampicillin 50μg/mL (E. coli Bl21(DE3) strain with Alpha casein and Ptac-SR-GST
Figure 1b: LB agar plate with Ampicillin 50μg/mL (E. coli Bl21(DE3) strain with mfp5-Alpha casein and CBD-Alpha
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.
- Conduct overnight culture of a single positive colony from each of our recombinant E.coli strains.
- Extract DNA from the solution.
- 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.
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.
3a) BBa_K3503001 (Alpha Casesin)
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.
4a) Coomassie blue staining
4b) Western blot
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).
5a) Coomassie blue staining
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).
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", 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" 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 our project.
Figure 7a.Placed the bed sheets in trays and soaked the bed sheets with different proteins
Figure 7b.Let the bed sheets dry overnight
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.
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 )
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.
Figure 9. Fire retardant test for wood
Before test
The wood smoldered
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.
10a)The percentage of the protein after processes
10b)RFP-Alpha Casein(BBa_K3503009) by different state of process(non- washing, after washing with water and soaking.)
10c)RFP-mfp5-Alpha casein(BBa_K3503011) by different state of process(non- washing, after washing with water and soaking.)
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.
(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 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
[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.