Engineering & Results
DBTL #1
Design
The purpose of this project is to confer flame retardancy to plants and other cellulose substrates. To do this we need to identify natural flame retardants and identify strong cellulose binding domains.
To start our project we started with the flame retardant proteins. Our research revealed three main characteristics that a flame retardant material must have in order to make mechanisms for protein flame retardants to work. An acid source, a carbon source and a blowing agent which releases large quantities of gases (1). A material with these characteristics is called an intumescent material. These act to form an insulating char which acts to prevent reigninition. It may burn first but then will not reignite, or will burn far slower (2). Another method is to release water or non-flammable gases while burning, such as nitrogen or other inert gases. This will act to either fully extinguish the flame or again prevent the spread of the fire.
To expand a bit as the protein heats up it begins to degrade. As it degrades gases such as nitrogen are released from the structure of the protein and into the surrounding area. This deprives the area of oxygen and contributes to the formation of a char layer. Proteins are also very good at forming a char layer due to their polymeric properties. This char layer protects materials below it and also prevents the fire from spreading. Flame retardants high in phosphorus and nitrogen have been shown to be the best performing systems (3).
While many proteins could potentially satisfy these requirements, certain proteins such as Caseins, Glutens, Soy Proteins and Gelatin have been identified as being good flame retardants (3) When exposed to heat Caseins form an expanded carbon structure filled with bubbles which prevents and limits the spread of the fire (4). Additionally, Caseins are abundant in glutamines which contain -NH2 groups, which are primed to produce nitrogen gas to form that inhibitory char (5).
Beyond caseins, Whey proteins are another type of protein that have been identified as potentially having strong flame retardant properties. This is because the even distribution of hydrophilic amino acids allows the proteins to have fantastic water absorbent properties, additionally whey proteins can form a strong gas barrier (5). Beta-lactoglobin is the primary component of whey proteins. Finally we found that soy is able to form an oxygen-limiting char which slows a flame down (6).
During the course of our research we discovered that DNA is also an incredible flame retardant. As mentioned previously flame retardants high in phosphorus and nitrogen have been shown to be the most effective. Additionally DNA contains all the necessary components of an intumescent system; the phosphate backbone acts as an acid, the deoxyribose acts as a carbon source and then finally the bases contain a lot of nitrogen which act as a blowing agent.
While DNA is a strong flame retardant it does not stick to cotton. So, we will instead use proteins to bind to DNA to attach it to the cellulose (7). To bind our proteins we settled on PurA, which is a single stranded binding protein that binds to purine rich sequences (8). Finally, since it is important for our proteins to bind tightly together we decided to experiment with the streptavidin and biotin interaction by purchasing biotinylated nucleotides and linking Streptavidin to a cellulose binding domain. In the meantime while working on our cellulose binding domains we will also transform pET28a into DH5a and purify in large quantities in order to verify the flame retardancy of DNA. Thus after our research we had settled on five proteins to test the flame retardant properties:
- Alpha-S-Casein from Bos Tarus
- Beta-LactoGlobin from Bos Tarus
- Soy BeanGlutenin
- PurA from E.coli
- Streptavidin from Streptomyces Avidinii
Now that we have our flame retardant proteins we needed a way to attach our proteins to cellulose. We settled on the cellulosome complex. This consists of a cellulose binding domain and then a dockerin and a cohesion. While naturally for the digestion of cellulose they can be used to attach to cellulose (9). While there are multiple different types of cellulose binding domains, we settled on the cellulose binding domain from the bacteria Clostridium Thermocellum due to its low Ka value (10).
We decided to start with the cellulose binding domain from Clostridium Thermocellum and decided to create a fusion protein of CbpA conjugated to CohIII. Additionally we made an RFP-DocIII in order to quantitatively assess the binding capability of the different cellulose binding domains.
Build
In order to make our constructs we pulled sequences from FASTA and then codon optimized for production in E.coli. At the end of our design stages we had settled on five possible cellulose binding domains. Additionally we removed the N-terminal targeting sequence from Streptavidin as previous research had shown that the N-terminus interrupts the binding of Streptavidin to Biotin. Flame retardants were linked to a 6XHIS tag using a serine spacer. And then linked via the same serine spacer to a Dockerin. The cellulose binding domain from Clostridium Thermocellum was linked to a Cohesion from Clostridium Thermocellum using a Clostridium specific linker. Our constructs were then created through a combination of Gibson Assembly and ordering full plasmids from twist. Constructs created through Gibson Assembly were transformed into NEB 5-alpha before being identified via colony PCR and verified through full plasmid sequencing.
Test
For initial testing stages we started by transforming our test constructs into BL21DE3. We then grew up the strains containing the RFP-DocIII plasmid and induced them with 1mM IPTG. We started with the RFP-DocIII plasmid because it served as an easy test subject, because we assumed that inducing expression of the RFP would cause the culture to turn red. However, while our constructs grew we noticed a growth deficit in the induced culture, which suggested increased metabolic stress on the cells which could possibly suggest production of proteins. We did not see a red color.
Figure 1. Comparative expression of OD600 levels of induced vs. noninduced plasmids for Cellulose Binding Domain conjugated to a Cohesion (CBM) and Red Fluorescent Protein (RFP) conjugated to a DocIII
In order to fully probe this question we grew up our RFP conjugated to Dockerin as well as a regular RFP before streaking onto two plates, one containing IPTG and required Antibiotic and then one without antibiotic. These plates were then incubated overnight at 37C. Before being imaged in the morning. The image in the morning showed that the regular RFP cultures turned red in the presence of the IPTG. But the ones containing the RFP_Doc did not turn red in presence of the IPTG.
Figure 2. Bacterial growth plates containing RFP_DocIII and unconjugated RFP. Plated both in presence or absence of IPTG
Suggesting that the dockerin was preventing the RFP from being successfully expressed in the cell, and after further research we found this is in fact the case (11). Interestingly, months later, we noticed that a pellet from an induced culture of the strain containing RFP_DocIII had turned red. Even though it was not red to begin with. Given that insoluble proteins can slowly leak out of E.coli cells, this could suggest that the reason for the lack of color from the RFP_DocIII could be that the protein is insoluble. When we attempted to purify the pET28a we got very low yields.
Learn
Protein: These two results suggested that our fusion proteins attached to a dockerin would not express in either an easy or efficient manner, so it was thus necessary to redesign our product. For the next iteration we would thus design our proteins without the dockerin. DNA: Although we observed differences in the burning time between the treated and untreated samples, weighing the samples before burning revealed that our drying time was far too short and so it was necessary to prep our samples a full day ahead of when we wanted to burn. We also decided to create a furnace with a live temperature readout in order to get more accurate results. Additionally in order to get more material we decided to transform a higher copy number plasmid into the cells.
DBTL #2
Design
Our design stayed essentially the same, all we did was remove the dockerins from the flame retardant candidates. The flame retardant candidates were linked to a 6xHIS tag to aid in purification before being linked to a cellulose binding domain via a serine spacer. Additionally, for this iteration we elected to drop the optimized Ribosome binding site in favor of a regular pET Ribosome Binding Site. This is to reduce complexity and ensure uniform expression.
Build
Our constructs were assembled using the optimized sequences. With the same spacers as before. We then ordered optimized sequences from Twist in a full plasmid format and transformed the full plasmid into a BL21DE3 expression strain.
Test
For the testing of our new parts we first started with verifying expressions. We did this by performing a protein growth curve on the samples. However, as you can see from the gel below, it was difficult to determine if there was any protein being expressed.
Figure 3. Initial SDS PAGE with ambiguous results
After running multiple SDS-Page gels in this format we decided to run a colony PCR on our transformed strains to ensure that the DNA was in fact in our cells.
Figure 4. Gel from the colony PCR on our strains containing our samples. From left: Ladder, Alpha-S-Casien, Alpha-S-Casein, Streptavidin-1, Streptavidin-2, SBG-1, SBG-2, PurA-1, PurA-2, pET28a-1, pET28a-2
Finally we realized that it was necessary to normalize to a standard OD600 value of 10 when we prepped our samples for the SDS-Page Gels. This prevents the SDS from creating big streaks.
Figure 5. SDS-Page gel using samples from growing our samples Alpha-S-Casein_CBM and BetaLactoglobin_CBM. From Left: BetaLactoglobin_CBM Not Induced, BetaLactoglobin_CBM 1 Hr post induction, BetaLactoglobin_CBM 2 HR post induction, BetaLactoglobin_CBM 3 Hr Post induction, Alpha-S-Casein_CBM 1 HR post induction, Alpha-S-Casein_CBM 2 HR post induction, 3 Hr Post induction, Alpha-S-Casein_CBM NI 3 HR post Induction (Different day), Alpha-S-Casein_CBM I 3 HR post Inducton (Differnt day), BetaLactoglobin_CBM NI 3HR post induction (Differnt day), BetaLactoglobin_CBM I 3HR post induction (Differnt day), Purified Alpha-S-Casein_CBM, Purified Betalactoglobin_CBM, BetaLactoglobin_CBM only soluable, Alpha-S-Casein_CBM only soluable. From this we can visualize expression of the BetaLactoglobin, and also confirm that the betalactoglobin is soluable.
Figure 6. SDS-Page gel using samples obtain from growing PurA_CBM and two other e.coli strains. From Left: DH5a 1 HR, DH5a 2 HR, DH5a 3 HR, DH5a 4 HR, DH5a 4HR, MG1655 1 HR, MG1655 2 HR, MG1655 3 HR, MG1655 3 HR, MG1655 3 HR, MG1655 4 HR, PurA_CBM 1 HR (Induced), PurA_CBM 3 HR (Induced) PurA_CBM 3 HR (Induced and spun). From this we may be able to confirm expression of the PurA_CBM complex, there is a distinct band in the 3 HR sample that is not present in the 1 HR sample. This also suggests that PurA_CBM is soluable in E.coli which is encouraging for ease of expression.
Once this was implemented we were able to confirm the expression of the Beta-Lactoglobin CbpA construct. However, it appears that our Alpha-S-Casein is not expressing. Additionally, as evidenced by the two lanes that contain the purified protein, it appears that our purification protocol needs modification.
DNA: We tested three different conditions, 0.625% w/v DNA, 1.25% w/v DNA and 2.5% w/v DNA. Upon testing of our samples we observed that the 2.5% w/v sample was able to resist burning at 400C for three minutes.
Learn
For future steps we will be sure to run all SDS-Page gels using the similar format as above, including normalizing to a specific OD600 in order to prevent SDS failure. Additionally we would like to perform iterative design on our linkers in order to optimize for expression in E.coli.
Testing Flameretardancy Round 1
Testing Flame Retardancy Round 1
This experiment investigated the effect of DNA treatment on the flammability of cotton samples using a furnace-based burning test. Two conditions were tested: untreated cotton (0% w/v DNA) and cotton treated with a 2.5% w/v DNA solution. Each sample was weighed before and after burning to determine mass loss and observed during combustion to record temperature and burning behavior. The untreated cotton ignited and burned rapidly, fully combusting within about 80 seconds, while the DNA-treated cotton exhibited delayed ignition, slower burning, and greater structural integrity, even after five minutes in the furnace. These results suggest that DNA treatment enhances flame resistance and thermal stability, indicating its potential as a bio-based flame retardant. However, more testing needs to be done in order to confirm these results and control the temperature measurements will help quantify performance more precisely.
Conditions Tested
| Condition Number | Water (uL) | DNA (uL) | % W/V DNA |
|---|---|---|---|
| 1 | 560uL | 0 | 0 |
| 2 | 452.2 | 107.8 | 2.5 |
Table 1: DNA coating conditions
Table 1 shows the DNA concentrations for our 2 conditions. Condition 1 was only water with 0% w/v DNA. Condition 2 was 2.5% w/v DNA. The solutions were dotted onto their respective pieces of cotton with a pipet.
Mass of cotton before and after burning
Note: After taking initial measurements the cotton was cut in half in order to fit in the furnace
Conditions Tested
| Condition Number | Mass before Burning (g) | Mass after Burning (g) | % of Cotton that Remains | % of Cotton that burned away | Notes |
|---|---|---|---|---|---|
| 1 | 0.2461 | 0.0176 | 7.15 | 92.85 | 1 piece was completely burnt away, so the mass after burning is just 1 burned piece |
| 2 | 0.2630 | 0.0236 | 8.97 | 91.03 | 1 piece was completely burned away, so the mass after burning is just 1 piece |
Table 2. Mass of Cotton for each condition before and afer burning
The mass of the cotton sample for each condition was recorded before and after burning them in the furnace. Initially, each cotton sample was found to be too large to fit on the furnace stand, so each was cut approximately in half, resulting in two samples per condition. The first sample for each condition was left in the furnace until it was completely combusted, while the second sample was removed once it had turned entirely black to measure the remaining mass after partial burning. The post-burning mass for Sample #2 was recorded for both conditions. For Condition 1, 92.85% of the cotton (Sample #1 and #2) mass was lost, while for Condition 2, 91.03% of the cotton (Sample #1 and #2) mass was lost. Table 2 shows the initial and final masses for each condition and percent of cotton that remained and burned away after the experiment.
In the spirit of DBTL, we relized the burning until complete incereration resulted in results that were difficult to compare between eachother. Additionally we realized that the burning was performed at different starting temperatures which again made the data difficult to compare between
For the next iteration, we plan to measure the mass of each sample before and after a controlled burn, and then limiting the furnace exposure to exactly one minute. This approach will allow us to compare mass loss across samples without complete combustion.
Results from Burning videos
Since the cotton was cut in half, each condition was tested twice, with four total tests conducted on the cotton samples. Sample #1 of Condition 1 and Sample #1 of Condition 2 both burned away completely. Table 3 shows the results of the experiment.
| Sample Activities | Condition 1, Sample #1 | Condition 2, Sample #1 |
|---|---|---|
| inserted | 410C at 0 seconds | 420C at 0 seconds |
| starts browning | 410C at 2 seconds | 428C at 2 seconds |
| Black | 425C at 25 seconds | 400C at 50 seconds |
| Smoking | 417C at 20 seconds | 400C at 50 seconds |
| Embers | 435C at 40 seconds | 397C at 60 seconds |
| Burned Away | 429C at 80 seconds | 419C at 100 seconds |
Table 3. Comparison of Samples #1 of the untreated (Condition 1) and DNA-treated (Condition 2) vegetation samples under equivalent furnace conditions
Sample #2 of Condition 1 and Sample #2 of Condition 2 were both taken out after burning to determine their mass and calulate the mass which burned away
| Sample Activities | Condition 1, Sample #2 | Condition 2, Sample #2 |
|---|---|---|
| inserted | 370C at 0 seconds | 410C at 0 seconds |
| starts browning | 370C at 2 seconds | 408C at 25 seconds |
| Black | 386C at 40 seconds | 423C at 50 seconds |
| Smoking | 386C at 40 seconds | 413C at 120 seconds |
| Embers | N/A | N/A |
| Removed | 400C at 120 seconds | 430C at 300 seconds |
Table 4. Comparison of the second sample of the untreated and DNA-treated samples.
To assess the performance and the effect of DNA as a flame retardnat, we compared untreated plant samples (condition 1) to treated ones (Condition 2). Howeverm because some samples were inserted into the furnance at different starting temperatures, only Condition1 Sample #1 and Condition 2 sample #2 were compared to ensure consistency across datasets. Both samples were introduced into the furnace at approximately 410°C, with temperature fluctuations remaining within 410°C–430°C throughout the trials. Table 5 below shows a comparison of the aforementioned conditions.
| Sample Activities | Condition 1, Sample #1 | Condition 2, Sample #2 |
|---|---|---|
| inserted | 410C at 0 seconds | 410C at 0 seconds |
| starts browning | 410C at 2 seconds | 408C at 25 seconds |
| Black | 425C at 25 seconds | 423C at 50 seconds |
| Smoking | 417C at 20 seconds | 413C at 120 seconds |
| Embers | 435 at 40 seconds | N/A |
| Removed | 429C at 80 seconds | 430C at 300 seconds |
Table 5. Comparison of untreated (Condition 1) and protein-treated (Condition 2) vegetation samples under equivalent furnace conditions.
The untreated sample (Condition 1) exhibited rapid combustion behavior: visible browning occurred within 2 seconds of insertion, followed by blackening and smoke production within 25 seconds. Embers appeared shortly after, and the sample was fully consumed in approximately 80 seconds.
In contrast, the protein-treated sample (Condition 2) showed significantly delayed combustion. Browning did not begin until 25 seconds, and visible smoke was not observed until around 120 seconds. Notably, the treated sample did not produce embers, retained structural integrity throughout, and required removal after five minutes at similar temperatures due to minimal burning.
These results demonstrate that the DNA substantially increased the sample’s resistance to ignition and thermal degradation. The delayed onset of browning and smoke, along with the absence of embers, indicates improved thermal stability and reduced flammability. Moving forward, repeating these trials with additional replicates and temperature-controlled measurements will help quantify performance more precisely. Nonetheless, this experiment highlights the furnace’s effectiveness as a reliable testing platform and validates the protein’s potential to enhance vegetation fire resistance.
Finally for next time, in addition to burning for a set time and ensuring consistent start temperature we will also work to fully submerge our cotton in the DNA solution. Our current protocol involves dotting the solution on with a pipette tip which could lead to inconsistent spreading of the sample and control, and this may explain the spotty results
References
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