iGEM 2022 — Team seoul-korea

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## Notebook

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## Engineering Success

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For our final goal of producing natural sorbents, we attempted to selectively degrade cellulose, a hydrophilic substance, by creating genetically engineered bacteria that can secrete cellulose-degrading enzymes. We had to let the enzyme produced by the bacteria be secreted to simplify the degradation process. However, since it is impossible to confirm all the previously known secretion signals, 12 secretion signals were selected by referring to the literature search and the results of TU\_Dresden who participated in iGEM in 2017. A signal peptide library plasmid was designed so that 12 secretion signals were selected for the coding sequence of each enzyme and cut into NdeI and NcoI for use.

![](https://static.igem.wiki/teams/4317/wiki/eng1.png)

[Figure 1. Plasmid map of signal peptide library]

We did not digest the plasmid DNA directly with NdeI or NcoI, but cut the library part after amplification using the primers (AIO F- GGATAACCGTATTACCGCCTTTGAG, AIO R- CACAAACAGACGATAACGGCTCTC) shown in Fig. 1 (Fig. 2, 3). Secretion signal fragments were ligated with the DNA fragments coding for each enzyme with Bacillus and E. coli expression vectors and transformed (Fig. 4).

![](https://static.igem.wiki/teams/4317/wiki/eng2.png)

[Figure 2. PCR product containing the signal peptide library]

![](https://static.igem.wiki/teams/4317/wiki/eng3.png)

[Figure 3. secretion signal fragments digested by NdeI, NcoI]

![](https://static.igem.wiki/teams/4317/wiki/eng4.png)

[Figure 4. Transformation result of plasmid ligated with signal peptide library and cellulases]

The enzymes overexpressed from the plasmid obtained through the screening process were actually used to decompose cellulose. In addition, we demonstrated that it can be used as an oil adsorbent by decomposing cellulose in pine powder using our decomposition device (Fig. 5, 6).

![](https://static.igem.wiki/teams/4317/wiki/hard1.png)

[Figure 5. Cellulose Decomposer]

![](https://static.igem.wiki/teams/4317/wiki/eng5.png)

[Figure 6. The color change of pine tree powder after 12 weeks of reaction.]

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## Experiment

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#### Design

![](https://static.igem.wiki/teams/4317/wiki/ex5.png)

[Fig. 1 4 Types of Enzymes related to Cellulose Decomposition]

Team Seoul\_Korea assured that the cellulose amount in decomposed pine tree bark decreases and that it can successfully and effectively absorb harmful oil. However, waiting for dissolution by microbes takes way too long and the outcome is far less than the amount needed to absorb oil. Also, as human labor is needed to find dissolved oil, the method takes up too much resources and time. Therefore, Seoul\_Korea aimed to produce a sorbent using pine trees with synthetic biology. First, by examining documents, Seoul\_Korea found 4 kinds of cellulose degrading enzymes (Cellobiohydrolase I, II, beta-glucosidase, endoglucanase) that have originated from bacteria or heterologous expression checked. (Figure 2). Also, to simplify the process of cellulose dissolving, Seoul\_Korea designed the experiment for signal peptides to fusion so that the enzymes could secrete. Seoul\_Korea randomly inserted 12 kinds of secretion signals and effectively found out the secretion signals of secretied clones by dot blot method to find the most suitable signal peptide of each enzyme. Seoul\_Korea applied the IPTG inducible system to express mass amounts of enzymes in E. coli and Bacillus subtilis. Through this process, enzymes secreted by media effectively dissolved cellulose, and Seoul\_Korea confirmed that using this method, it takes 1/10 of the time to make a sorbent from our hardware system compared to nature producing the same product.

#### Protocols

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## Results

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![ ](https://static.igem.wiki/teams/4317/wiki/res1.png)

[Fig. 1. Cellulose degrading enzymes]

#### Par I

Through literature and database search, we chose enzymes that could break down cellulose in pinewood, such as cellobiohydrolase I (KsCel7A) from Kocuria palustris, cellobiohydrolase II (TfCel6B) from Thermobifida fusca, synthesized based on ancestral endocellulase (LFCA\_EG - Cel5A), and beta-glucosidase (CtBglA) from Clostridium thermocellum. These enzymes have high activity levels and can be epxressed from E. coli and B. subtilis. We predicted the tertiary structure of these enzymes using Alphafold. We found that in all proteins except CtBglA, the catalytic part and the carbohydrate-binding module formed an intact structure. Also, CtBglA could exist in a soluble form to effectively decompose cellobiose released from cellulose by CBHI, II, and LFCA-EG.

![](https://static.igem.wiki/teams/4317/wiki/res2.png)

[Fig. 2. Transcriptional activity of various promoters in Bacillus subtilis]

We tested several types of promoters to express the cellulose-degrading enzyme in Bacillus subtilis and Escherichia coli. Through literature review, we selected four constitutive promoters and two inducible promoters known to be strong in Bacillus. To confirm that these promoters are working properly, mScarlet was used as a reporter. As shown in Figure 2, trnQ showed the strongest transcriptional activity. In addition, transcription by the grac100 promoter was inhibited by Lacl.

![](https://static.igem.wiki/teams/4317/wiki/res3.png)

[Fig. 3. IPTG inducible expression system]

For mass production of enzymes, we checked whether the IPTG inducible system in bacillus works normally. The bacillus having the plasmid DNA shown in Figure 3 was spread on an LB agar plate with or without IPTG. As a result, it was confirmed that the expression of the reporter gene was high in the condition with IPTG. These results show that our IPTG inducible system works normally in Bacillus subtilis.

![](https://static.igem.wiki/teams/4317/wiki/res4.png)

[Fig. 4. Transcriptional activity of various promoters in Escherichia coli]

On the other hand, the results were different in E. coli. The transcriptional activity of the P2069M promoter was higher than that of the trnQ promoter. As in Bacillus, the grac100 promoter was effectively inhibited by Lacl, but IPTG did not induce the expression of the reporter gene. Therefore, we used the previously known lac promoter of E. coli for production of cellulose degrading enzymes.

We tried to decompose cellulose by directly treating the media containing the enzymes on wood by simplifying the complex process of 'expression, purification, and decomposition' of the cellulose degrading enzyme and 'expression and secretion'. To this end, it was attempted to find a secretion signal through which each enzyme could be effectively secreted. A signal peptide library was prepared by cutting sp-library plasmid containing 12 secretion signals with NdeI and NcoI. The coding sequence of each enzyme was cut and purified with NcoI and XhoI, and PtrnQ and P2069M plasmids were prepared by cutting with NcoI and XhoI. Four ligation samples using PtrnQ as a backbone were transformed into Bacillus subtilis 1A976, and four recombinant DNAs using P2069M plasmid as a vector were transformed into E. coli Mach1. Transformants were spread on agar medium containing chloramphenicol and ampicillin, respectively. Each of 24 colonies was inoculated in a 96 well plate and incubated at 37°C for 12 hours. After centrifugation, the supernatant was spotted on a nitrocellulose membrane. After immunoblotting with an anti-his tag antibody, two clones showing a strong signal were selected and their plasmid DNA was isolated. Then, the secretion signal was identified through sequencing.

![](https://static.igem.wiki/teams/4317/wiki/res5.png)

[Fig. 5. Screening of secretion signals of KsCel7A]

The secretion of KsCeli7A by epr and ywsB was the highest. Bacillus was most effectively secreted by bglC and ywsB.

![](https://static.igem.wiki/teams/4317/wiki/res6.png)

[Fig. 6. Screening of secretion signals of TfCel6B]

In the case of KsCel7A, E. coli was secreted the highest by epr and ywsB. Bacillus was effectively secreted by bglC and ywsB.

![](https://static.igem.wiki/teams/4317/wiki/res7.png)

[Fig. 7. Screening of secretion signals of LFCA\_EG]

LFCA\_EG was enhanced by pel in both Escherichia coli and Bacillus subtilis, and also helped to secrete bglC and aprE.

![](https://static.igem.wiki/teams/4317/wiki/res8.png)

[Fig. 8. Screening of secretion signals of CtBglA]

Lastly, for CtBglA, phoB was the most frequent, and ywsB was observed in the rest.

The combination of 16 secretion signals and enzymes found previously was subcloned using the IPTG inducible system. These plasmids were transformed into Bacillus 1A976 and E. coli BL21 and filtered with a 0.2um CA syringe filter after induction with IPTG. Each sample was separated by size through SDS-PAGE. The resolved protein was transferred to a 0.45um PVDF membrane and then immunoblotted with an anti-his tag antibody. Finally, the blotted membrane was stained with amido black.

![](https://static.igem.wiki/teams/4317/wiki/res9.png)

[Fig. 9. SDS-PAGE and western blot of cellulose-degrading enzymes secreted from B. subtilis and E. coli]

It was found that SP\_ywsB-TfCel6B and SP\_bglC-LFCA\_EG were effectively expressed and secreted in BL21. Although the overall expression level was low in 1A976, SP\_phoB-CtBglA and SP\_ywsB-KsCel7A were stably expressed.

![](https://static.igem.wiki/teams/4317/wiki/res10.png)

[Fig. 10. Clear zone on CMC agar plates after staining with congo red]

The activity of LFCA\_EG was confirmed further in the CMC agar plate. It was observed that LFCA\_EG secreted from both BL21 and 1A976 effectively decomposed CMC. However, according to the previous SDS-PAGE results, LFCA\_EG proteins of the desired size were more present in BL21. Therefore, we used SP\_bglC-LFCA\_EG produced from BL21.Ultimately, we combined SP\_ywsB-TfCel6B and SP\_bglC-LFCA\_EG produced in BL21 and SP\_ywsB-KsCel7A produced in 1A976 to break down the cellulose.

![](https://static.igem.wiki/teams/4317/wiki/res11.png)

[Fig. 11. Cellulose degradation by treatment with mixtures of secreted enzymes]

We overexpressed the aforementioned plasmid in the E.coli BL21 and B. subtilis 1A976 for 12 hours at 30 degrees in the 0.4mM IPTG condition. After centrifugation, we obtained a supernatant containing the enzyme. Without purification, we mixed the combination mentioned in the figure 11, treated it with cellulose, and measured the amount of reducing sugar through the DNSA assay. As a result, KsCel7A failed to contribute much to the breakdown of cellulose. We thought this was due to the small amount of protein secreted. However, treating the 4 enzymes together resulted in the greatest amount of cellulose broken down. Therefore, we decided to use a mixture of the four enzymes

![](https://static.igem.wiki/teams/4317/wiki/res12.png)

[Fig. 12. Determination of maximal activity time of cellulose-degrading enzyme cocktails]

Additionally, we observed the degree of degradation over time to determine how long our cellulose degrading enzyme cocktail functions. As time passed, the activity of the enzyme gradually decreased, but cellulose was maximally degraded between 18 and 24 hours after the enzyme treatment. After 24 hours, the amount of glucose in the solution starts to decrease, which is thought to be the effect of the growth of bacteria.

##### Future Work

Our team has confirmed that it is possible to produce oil adsorbents by effectively decomposing cellulose in wood through experiments so far. However, the low expression and secretion of KsCel7A with cellobiohydrolase I activity is a problem to be solved. So, we cloned the coding sequence of KsCel7A into the pPICZalpha plasmid to confirm that a higher amount of protein can be obtained from KsCel7A in Pichia pastoris. In addition, in order to easily isolate positive clones from Pichia, T2A-self-cleaving peptide and sfGFP were fused behind the coding sequence of KsCel7A. When the KsCel7-T2A-sfGFP fusion construct is transcribed and translated by the AOX1 promoter to produce a long peptide, it is cleaved into KsCel7A and sfGFP by T2A. Therefore, positive clones can be easily distinguished by the presence or absence of fluorescence. In Figure 13, the plasmid was electroporated to pichia after linearization with PmeI. 20 colonies were patched on a minimal methanol plate containing methanol. As a result, two clones showing high fluorescence intensity were obtained. We plan to measure secretion and enzyme activity of KsCel7A by inducing overexpression of KsCel7A in Pichia.

![](https://static.igem.wiki/teams/4317/wiki/res13.png)

[Fig. 13. Pichia secretion plasmid and Positive clone of pichia expressing KsCel7A]

#### Part II

##### Cellulose degradation in pine tree

![](https://static.igem.wiki/teams/4317/wiki/res14.png)

[Fig. 14. The color change of pine tree powder after 12 weeks of reaction.]

For the effective elimination of cellulose from pine woods, we pulverized the wood waste to make it easier for enzymes to react. After decomposing pine tree powder using four types of enzymes for twelve weeks through our self-made reaction machine, we observed that the color of the powder turned darker brown compared to that before the reaction. Although there was a slight difference in color and brightness depending on the types of pine tree powder samples, the overall trend shows that the color of the powder got darker throughout the twelve weeks of reaction.

##### Contact Angle Analysis

![](https://static.igem.wiki/teams/4317/wiki/res15.png)

[Fig. 15. Contact Angle Analysis of pine tree powder]

In our pilot experiment, we observed the increase of hydrophobicity of pine wood powder that is decomposed by brown rot fungi when it is heated at 200℃ for 10 minutes. Also, since we know that the degree of hydrophobicity is directly proportional to the lignin composition of the powder, we attempted to increase the amount of lignin by using four types of enzymes that decompose cellulose from pine tree powder. To see if reaction time affects the hydrophobicity level, we collected samples from the week 1, 4, and 8 weeks and measure the contact angle. As a result, the contact angle of heated samples increases as reaction time gets longer. Although we couldn’t collect data from a longer time period due to the number limit of the reaction machine, there wasn’t a noticeable difference between weeks 12 and 15.

##### Change in Hydrophobicity Duration Time with Regards to Lignin Content

![](https://static.igem.wiki/teams/4317/wiki/res16.png)

[Fig. 16. Change in Hydrophobicity Duration Time with Regards to Lignin Content]

Here, we released five grams of pine wood powder that is heated at 200℃ and reacted with four types of enzymes on the water and measure the time it took for the powder to start sinking down. As we know that the longer reaction time with enzymes increases the lignin composition of the powder, it was reasonable to see that the lignin content of the initial sample increased from 27.5% to 45.6% on through the twelve weeks of reaction. Another interesting aspect of this experiment was that the duration of hydrophobicity also increased as the powder reacted with enzymes longer. In our observation, there was a huge increase in the duration of hydrophobicity after the eight weeks of reactions, and when the twelve weeks of reaction was completed, the sample floated on the water for 26 hours, which is three times as longer than the duration in week 8. While the lignin composition showed linear growth, the duration of hydrophobicity showed exponential growth throughout the twelve weeks of observation. Since the lignin content was 45.6% at the end of the experiment, Wwe concluded that the hydrophobicity of the pine wood powder increases significantly once it gets over 40% of the lignin composition. Therefore, we determined twelve weeks of reaction time to be the most effective time interval to induce hydrophobicity and named the pine tree powder from this reaction as “PineSorb”.

##### Hydrophobicity & Oleophilicity

![](https://static.igem.wiki/teams/4317/wiki/res17.png)

[Fig. 17. Hydrophobicity & Oleophilicity of Pinesorb]

To test the hydrophobicity and oleophilicity of PineSorb after twelve weeks of reaction time, we put PineSorb in a circle, flattened the surface using a wooden plank, and reacted with water and different types of solutions. While water contacted with PineSorb and formed circular waterdrops at the surface, other solutions that are relatively non-polar were absorbed extremely well by PineSorb.

![](https://static.igem.wiki/teams/4317/wiki/oil2.gif)
![](https://static.igem.wiki/teams/4317/wiki/prof4.gif)

After releasing the mixture of diesel oil and Nile Red dye and putting a tea bag with 4g of PineSorb, we can observe that Pinesorb absorbs almost every oil on the water in a short period of time.

After putting a beaker of water on the stirrer and releasing waste engine oil, we put 4g of PineSorb and started the stirrer. Despite the strong water current, the tea bag with PineSorb did not sink down and removes almost every waste engine oil on the water's surface. From this experiment, we can learn that PineSorb sustains its high hydrophobicity while absorbing oil efficiently.

![](https://static.igem.wiki/teams/4317/wiki/oil.gif)

In the water tank that imitates actual environments like oceans or lakes, we filled in water, released waste engine oil, and scattered the PineSorb powder. After a very short period of time, we pushed the powder with a stick and observed how it eliminates oil by forming a thin layer on the surface. Since the powder does not sink even after adsorbing oil, it is possible to completely remove spilled oil from the water.

##### Conclusion

![](https://static.igem.wiki/teams/4317/wiki/res18.png)

When we activated four types of enzymes that decompose cellulose at two types of bacteria—Bacillus subtilis and Escherichia coli—and treated pine tree powder, it effectively decomposed cellulose through twelve weeks and increased lignin composition from 25~30% to 39~47%. The amount of time this process has taken is extremely shorter than when a pine tree is decomposed by brown rot fungi in a natural environment. Although we did not directly compare the same exact samples in our experiments, the fact that we increased the hydrophobicity and oleophilicity of pine trees in such a short period of time has enough value and merchantability in the eco-friendly oil adsorbent market. Based on this achievement we made through experiments, we are optimistic that we can even shorten the time it takes for pine wood to be decomposed by utilizing a fungi system and decomposing hemicellulose with cellulose together. Lastly, I believe that this study opened more possibilities for the use of lignin for industrial purposes.

## References

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1) Noh, J. H., Kim, D. S., and Sung, Y. J.(2017). The isolation of kraft lignin from black liquor during Korean red pine kraft pulping and evaluation of the isolated kraft lignin, Journal of Korea TAPPI 49(4):170-177

2) Traoré, M., Kaal, J., & Cortizas, A. M. (2018). Differentiation between pine woods according to species and growing location using FTIR-ATR. Wood science and technology, 52(2), 487-504.

3) Kim, C.K., Chun, J.Y., Shin, Y.S., Hwang, S.I., Kang, B., et al., (2017), A Preemptive Study on Marine Pollution Response Caused by Environmental Changes such as International Ship Fuel Oil Regulation, Ministry of Public Safety and Security, Sejong, Republic of Korea, 1-112.

4) Kim, S. J., Kang, J., Youe, W. J., Lee, S. M., & Kim, Y. S. (2017). Preparation and mechanical properties of melt-spun carbon fibers from wood tar and wood tar/kraft lignin blends. Journal of Korea TAPPI, 49(5), 56-64.

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