iGEM 2022 — Team keystone

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}{% endblock %}
{% block page\_content %}

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering{% endblock %}
{% block lead %}Showcasing our engineering process{% endblock %}
{% block page\_content %}

# Abstract

The social dilemma of body odor has provoked a demand for the deodorant industry. Currently, chemical deodorants and surgery are the most prevalent solutions to body odor. However, due to the harm of chemical deodorants and high cost of surgery, KEYSTONE 2022 aims to create a safer and cheaper product for the effective mediation of body odor,which is our project AROMATA. AROMATA is mainly composed of fengycins from Bacillus subtilis 168 to inhibit the quorum sensing system of Staphylococcus spp., the producer of axillary malodor, thus decrease their detachment to skin and realize deodorization. Meanwhile, santalene from E. coli DH5α is also added into our product for the pleasant sandalwood aroma.By mixing fengycins and santalene, we have designed spray and hydrogel product for implementation. We sincerely hope that our product can solve the problem of body odor in a safer, more eco-friendly and more convenient approach to alleviate the social dilemmas faced by people, and then become a new trend in the cosmetic industry.

![](https://static.igem.wiki/teams/4274/wiki/figure-1.jpg)

Figure 1. Bacillus subtilis 168 will produce fengycins to bind with Staphylococcus spp., inhibit their quorum sensing, and thus decrease their detachment to skin. Escherichia coli DH5α will produce santalene to provide sandalwood aroma. By mixing fengycins and santalene, we can produce a series of AROMATA product, such as spray and hydrogel.

# Target 1 Fengycins production in Bacillus subtilis 168

The main composite of our product, fengycins, is produced by engineering Bacillus subtilis 168. As it is shown in the demonstration in description, sfp and degQ genes are critical to the biosynthesis of fengycins. Besides, Bacillus subtilis 168 possesses a natural but invalid sfp gene, and the functional degQ gene is silenced with the regulation of a weak promoter.Thus, with the help of pJOE8999\_sfp\_degQ plasmid (Figure 2a), we knocked in both sfp gene and degQ gene in the region of the natural sfp gene of Bacillus subtilis 168 after being knocking out. Under the guidance of protocol, we successfully transformed and induced the plasmid to function as a gene editor (Figure 2c). After induction, 7 strains were selected for PCR and electrophoresis verification. Since successful knock-in would would result in a 252 bps increase in the genome of Bacillus subtilis 168, the electrophoresis result conveys preliminarily that all of these strains has achieved our target of constructing the fengycins producers (Figure 2b).

![](https://static.igem.wiki/teams/4274/wiki/figure-2.jpg)

Figure 2. Edting the genome of Bacillus subtilis 168 to enable it to produce fengycins. (a) Plasmid design for knocking out the invalid sfp gene of Bacillus subtilis 168 and knocking in the sfp gene from B. amyloliquefaciens FZB42 and degQ gene from Bacillus subtilis 168. (b) Electrophoresis results show that gene editing is successful. (c) Protocol about transformation, induction and elimination of pJOE8999 plasmid in Bacillus subtilis 168.

After sequencing further verified that the successful construction of the fengycins producing strain, we used the strain for fermentation (Figure 3a). For the quantification of fengycins production, the methanol - extract obtained from the fermentation broth was analyzed by high performance liquid chromatography (HPLC). The peak of fengycins appeared at the retention time of 8-11.5 min under methanol mobile phase (Figure 3c). The injection sample was a solution of lipopeptide extract from 30 mL of fermentation broth dissolved in 7 mL methanol. Besides, various concentrations of fengycins standard is also injected for the construction of the standard curve of peak area obtained by HPLC (y) and its corresponding fengycins’ concentration (x). Based on the standard curve, we got the regression curve and its corresponding formula y=9.767x+193.44, and R2>0.98 displays that the regression equation fits the observed values (Figure 3b). Substituting y=1242.3 obtained from the sample into the regression equation, the concentration of fengycin in methanol solution can be calculated as 107.4 mg/L, that is, 25.06 mg/L of fengycins can be achieved by shaking-flask fermentation.

![](https://static.igem.wiki/teams/4274/wiki/figure-3.jpg)

Figure 3. Quantification analysis of fengycins production. (a) The sequencing result of engineering Bacillus subtilis 168 strain for production. (b) The standard curve of peak area obtained by HPLC (y) and its corresponding fengycins’ concentration (x). (c) HPLC results of sample and various concentrations of fengycins standard.

# Target 2 Fengycins’ inhibitory effect on the quorum sensing system of Staphylococcus hemolyticus

The deordoring function of our product is achieved by the inhibitory effect fengycin displays on the quorum sensing system of Staphylococcus spp. And the inhibitory effect of the product was verified by biofilm quantitative detection at different concentrations of lipopeptide extract. In this detection, the available Staphylococcus haemolyticus, one of the guilty pathogens to be blamed for body odor, is available in the lab of Nanshan Hospital and thus selected. The results demonstrated that the biofilm content of S. haemolyticus was significantly decreased under the treatment of 1.25 g/L and 2.5 g/L lipopeptide extract. In particular, 1.25 g/L extract (containing 5.625μM fengycins) resulted in a reduction of about 20% in biofilm content of S. haemolyticus. The experimental results are consistent with our hypothesis, which is most likely because fengycins compete with autoinducing-peptide (AIP) for the binding site of agr quorum sensing system, and then inhibits its biofilm formation. (Figure 4a)

In view of the current situation that the abuse of antibiotics leads to the emergence of drug-resistant bacteria, our project has the courage to be responsible for the better world, and thus fengycin, an antibiotic that inhibits bacteria by inhibiting the quorum sensing system, was chosen to be the core of our project. The measurement of the growth curve of S. hemolyticus at different concentrations of lipopeptide extract was just designed to verify that our lipopeptide extract will not kill the pathogens. As it is shown in Figure 4b, 1.25 g/L of extract did not inhibit S. hemolyticus growth before 15 hours, which was also consistent with our assumption (Figure 4b). It could be concluded that 1.25 g/L of lipopeptide extract (containing 5.625μM fengycins) would be the optimal inhibitory concentration to be applied in our product.

![](https://static.igem.wiki/teams/4274/wiki/figure-4j.jpg)

Figure 4. Fengycins’ inhibitory effect on the quorum sensing system of Staphylococcus haemolyticus. (a) Biofilm quantitative detection of Staphylococcus hemolyticus at different concentrations of lipopeptide extract. (b) Measurement of the growth curve of Staphylococcus hemolyticus at different concentrations of lipopeptide extract.

# Target 3 Santalene production in E.coli DH5α

As stated in the project description, after engineering, E. coli could utilize both MEP pathway and MVA pathway for the universal precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), then synthesize santalene with the help of FPP Synthase (FPPS) and santalene synthase (SS). Except heterologously expressed MVA pathway and ERG20 of Saccharomyces cerevisiae and santalene synthase of Clausena lansium (ClSS), several modifications upon ERG20 or ClSS by amino acid mutation, binding to a hydrophillic tag and the construction of fusion protein were tested for the higher yield of santalene. Therefore, with the help of the co-transformation of pMVA plasmid with various pW1 plasmids, including pW1\_CE, pW1\_CEM, pW1\_TCEM and pW1\_CEM\_FL, different strains like CE, CEM, TCEM, CEM\_FL were successfully constructed (Figure 5). The complete pathway we designed for producing santalene in E. coli is illustrated in Figure 5.

![](https://static.igem.wiki/teams/4274/wiki/figure-5.jpg)

Figure 5. Construction and expression of santalene. (a) Enzymes and some of the reaction intermediates necessary for the production of santalene through the MEP pathway and MVA pathway. (b) Schematic representing the structure of pMVA, pW1\_CE, pW1\_CEM, pW1\_TCEM and pW1\_CEM\_FL transformed into E.coli DH5α ∆TnaA.

Afterwards, the various engineering of E.coli DH5α ∆TnaA mentioned above were used for santalene production. After rapid centrifugation, the supernatant of dodecane was spiked with with 0.475 g/L a-humulene as an internal standard, and then injected into GC/MS for verification of α-santalene production. It turned out that all samples from four strains appeared a significant peak at the retention time of 26-27 min, and various peak area of different samples exhibited santalene production with differing levels, indicating the general success of E. coli engineering. It can be concluded that the E. coli strain CEM (with pW1\_CEM plasmid) produces the maximal level of α-santalene compared to other strains (73.93 mg/L). Furthermore, our study elucidates that the mutation of 96th amino acid into tryptophan could increase the yield of α-santalene by about 20%, substantiating the prominent performance of ERG20F96W in enhancing the supply of FPP and α-santalene production in E. coli (Figure 6).

![](https://static.igem.wiki/teams/4274/wiki/engineering/figures/figure6.png)

Figure 6. Quantification analysis of α-santalene production. (a) Measurement santalene production of different strains by GC/MS results. (b) Quantification of α-santalene is analyzed with 0.475 g/L α-humulene as an internal standard. And the GC/MS results demonstrate that the peaks at the retention time of 26-27 min and 28-29 min respectively were α-santalene and α-humulene. (c) GC/MS results of samples originated from CE, CEM, TCEM and CEM-FL strains.

# Conclusion and Discussion

Our team had successfully engineered Bacillus subtilis 168 to produce fengycins, capable of inhibiting staphylococcal population, and biosynthesized α-santalene from E.coli, promoting a synthetic fengycins- based deodorant with the scent of sandalwood was developed.

Due to related policy that impose the ban of living bacteria application on exposed human body, part of our project cannot be implemented fully. If the limitation of biological synthesized product alleviate, we would proceed with the engineering of live bacteria hydrogel products that modify Bacillus subtillis by using its dtpt in the absorption of Cys-Gly-3M3SH on skin, the precursor of 3M3SH odor molecules. Our team would also introduce the suicide mechanism to ensure the safety and enhance deodorant effect, meanwhile avoid the addition of environmental burden.

Currently, our team succeed in knocking out the patB enzyme in the Bacillus subtilis 168, guarantee that Bacillus subtilis 168 can both absorb Cys-Gly-3M3SH and avoid the production of odor. This result had laid the experimental foundation and pave the way for our second-generation product (Figure 7).

The possibilities opened up by the AROMATA are endless. As people with body odor is faced with a rather severe social dilemma and in lack of a safer, more eco-friendly and more convenient solution. The AROMATA hopes to be a better solution to body odor, and become a new trend in the cosmetic industry.

![](https://static.igem.wiki/teams/4274/wiki/engineering/figures/figure7.png)

Figure 7. Screening results of Bacillus subtilis strains with successful patB gene knock-out. (a) Electrophoresis results show that gene editing is successful. (b) The schematic representation of Bacillus subtilis genome before and after patB being knocked-out.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}{% endblock %}
{% block page\_content %}

## 1.Modification of Bacillus subtilis 168 to produce fengycins

The pJOE8999\_sfp\_degQ plasmid is obtained from the Suzhou Silicon based Biotechnology Co., Ltd (China). After the plasmid was transformed into Bacillus subtilis 168, the kanamycin resistant clones were selected for 0.2% mannose-induction. And the modified clones would be verified by colony PCR and sequencing. And the modified strain would be utilized for fengycins production. After that fengycins would be extracted from fermentation broth and then quantified by HPLC. (Tan et al, 2022)

In this section, composite part BBa\_K4274035 was used.

Visit the protocol below for more details about experimental procedures.

## 2.Modification of Escherichia coli DH5α to produce santalene

pW2 plasmid for the expression of ClSS and pW34 plasmid for the expression of ERG20 are obtained from the Suzhou Silicon based Biotechnology Co., Ltd (China). To construct plasmid pW1\_CE, pW1\_CEM and pW1\_TCEM, golden gate assembling and transformation were carried out in steps. Plasmid pW1\_CEM\_FL is constructed as the following experimental steps: PCR on pW1\_CEM plasmid, agarose gel electrophoresis, gel extraction, golden gate assembling and transformation. After verifying by colony PCR and sequencing, pW1\_CE, pW1\_CEM, pW1\_TCEM and pW1\_CEM\_FL were co-transformed with pMVA into Escherichia coli DH5α, then strain CE, CEM, TCEM, CEM\_FL were successfully constructed and utilized for santalene production. After rapid centrifugation, the supernatant of dodecane was spiked with with 0.475 g/L a-humulene as an internal standard, and then injected into GC/MS for quantification analysis of α-santalene production. (Jia Z. et al, 2022)

In this section, composite parts BBa\_K4274030, BBa\_K4274031, BBa\_K4274032 and BBa\_K4274033 were used.

Visit the protocol below for more details about experimental procedures.

## 3.Modification of Bacillus subtilis 168 to unable itself to produce 3M3SH

The pJOE8999\_patB plasmid is constructed followed by patB\_target assembling and homology arms assembling. The short DNA fragment patB\_target was obtained by T4 PNK phosphorylation of DNA oligos and then golden gate assembing with pJOE8999 plasmid. Homology arms for repairing were obtained by PCR amplification of Bacillus subtilis and gel extraction. After digestion by Bgl1 and ligation by T4 ligase with pJOE8999\_patB\_target, the pJOE8999\_patB plasmid was successfully constructed.
Same as pJOE8999\_sfp\_degQ plasmid, patB gene was knocked-out after transformation, induction and elimination. And colony PCR results could be used to prove the clones selected were modified.
In this section, composite part BBa\_K4274036 was used.
Visit the protocol below for more details about experimental procedures.

## Protocols

## Reference

[1]Tan W., Yin Y., Wen J. (2022). Increasing fengycin production by strengthening the fatty acid synthesis pathway and optimizing fermentation conditions. Biochemical Engineering Journal, 177: 108235. https://doi.org/10.1016/j.bej.2021.108235

[2]Jia Z., Xun W., Xinyi Z., et al.(2022) Sesquiterpene synthase engineering and targeted engineering of α-santalene overproduction in Escherichia coli. J. Agric. Food Chem., 70(17):5377-5385. https://pubs.acs.org/doi/10.1021/acs.jafc.2c00754

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering{% endblock %}
{% block lead %}Showcasing our engineering process{% endblock %}
{% block page\_content %}

# Abstract

The social dilemma of body odor has provoked a demand for the deodorant industry. Currently, chemical deodorants and surgery are the most prevalent solutions to body odor. However, due to the harm of chemical deodorants and high cost of surgery, KEYSTONE 2022 aims to create a safer and cheaper product for the effective mediation of body odor,which is our project AROMATA. AROMATA is mainly composed of fengycins from Bacillus subtilis 168 to inhibit the quorum sensing system of Staphylococcus spp., the producer of axillary malodor, thus decrease their detachment to skin and realize deodorization. Meanwhile, santalene from E. coli DH5α is also added into our product for the pleasant sandalwood aroma.By mixing fengycins and santalene, we have designed spray and hydrogel product for implementation. We sincerely hope that our product can solve the problem of body odor in a safer, more eco-friendly and more convenient approach to alleviate the social dilemmas faced by people, and then become a new trend in the cosmetic industry.

![](https://static.igem.wiki/teams/4274/wiki/figure-1.jpg)

Figure 1. Bacillus subtilis 168 will produce fengycins to bind with Staphylococcus spp., inhibit their quorum sensing, and thus decrease their detachment to skin. Escherichia coli DH5α will produce santalene to provide sandalwood aroma. By mixing fengycins and santalene, we can produce a series of AROMATA product, such as spray and hydrogel.

# Target 1 Fengycins production in Bacillus subtilis 168

The main composite of our product, fengycins, is produced by engineering Bacillus subtilis 168. As it is shown in the demonstration in description, sfp and degQ genes are critical to the biosynthesis of fengycins. Besides, Bacillus subtilis 168 possesses a natural but invalid sfp gene, and the functional degQ gene is silenced with the regulation of a weak promoter.Thus, with the help of pJOE8999\_sfp\_degQ plasmid (Figure 2a), we knocked in both sfp gene and degQ gene in the region of the natural sfp gene of Bacillus subtilis 168 after being knocking out. Under the guidance of protocol, we successfully transformed and induced the plasmid to function as a gene editor (Figure 2c). After induction, 7 strains were selected for PCR and electrophoresis verification. Since successful knock-in would would result in a 252 bps increase in the genome of Bacillus subtilis 168, the electrophoresis result conveys preliminarily that all of these strains has achieved our target of constructing the fengycins producers (Figure 2b).

![](https://static.igem.wiki/teams/4274/wiki/figure-2.jpg)

Figure 2. Edting the genome of Bacillus subtilis 168 to enable it to produce fengycins. (a) Plasmid design for knocking out the invalid sfp gene of Bacillus subtilis 168 and knocking in the sfp gene from B. amyloliquefaciens FZB42 and degQ gene from Bacillus subtilis 168. (b) Electrophoresis results show that gene editing is successful. (c) Protocol about transformation, induction and elimination of pJOE8999 plasmid in Bacillus subtilis 168.

After sequencing further verified that the successful construction of the fengycins producing strain, we used the strain for fermentation (Figure 3a). For the quantification of fengycins production, the methanol - extract obtained from the fermentation broth was analyzed by high performance liquid chromatography (HPLC). The peak of fengycins appeared at the retention time of 8-11.5 min under methanol mobile phase (Figure 3c). The injection sample was a solution of lipopeptide extract from 30 mL of fermentation broth dissolved in 7 mL methanol. Besides, various concentrations of fengycins standard is also injected for the construction of the standard curve of peak area obtained by HPLC (y) and its corresponding fengycins’ concentration (x). Based on the standard curve, we got the regression curve and its corresponding formula y=9.767x+193.44, and R2>0.98 displays that the regression equation fits the observed values (Figure 3b). Substituting y=1242.3 obtained from the sample into the regression equation, the concentration of fengycin in methanol solution can be calculated as 107.4 mg/L, that is, 25.06 mg/L of fengycins can be achieved by shaking-flask fermentation.

![](https://static.igem.wiki/teams/4274/wiki/figure-3.jpg)

Figure 3. Quantification analysis of fengycins production. (a) The sequencing result of engineering Bacillus subtilis 168 strain for production. (b) The standard curve of peak area obtained by HPLC (y) and its corresponding fengycins’ concentration (x). (c) HPLC results of sample and various concentrations of fengycins standard.

# Target 2 Fengycins’ inhibitory effect on the quorum sensing system of Staphylococcus hemolyticus

The deordoring function of our product is achieved by the inhibitory effect fengycin displays on the quorum sensing system of Staphylococcus spp. And the inhibitory effect of the product was verified by biofilm quantitative detection at different concentrations of lipopeptide extract. In this detection, the available Staphylococcus haemolyticus, one of the guilty pathogens to be blamed for body odor, is available in the lab of Nanshan Hospital and thus selected. The results demonstrated that the biofilm content of S. haemolyticus was significantly decreased under the treatment of 1.25 g/L and 2.5 g/L lipopeptide extract. In particular, 1.25 g/L extract (containing 5.625μM fengycins) resulted in a reduction of about 20% in biofilm content of S. haemolyticus. The experimental results are consistent with our hypothesis, which is most likely because fengycins compete with autoinducing-peptide (AIP) for the binding site of agr quorum sensing system, and then inhibits its biofilm formation. (Figure 4a)

In view of the current situation that the abuse of antibiotics leads to the emergence of drug-resistant bacteria, our project has the courage to be responsible for the better world, and thus fengycin, an antibiotic that inhibits bacteria by inhibiting the quorum sensing system, was chosen to be the core of our project. The measurement of the growth curve of S. hemolyticus at different concentrations of lipopeptide extract was just designed to verify that our lipopeptide extract will not kill the pathogens. As it is shown in Figure 4b, 1.25 g/L of extract did not inhibit S. hemolyticus growth before 15 hours, which was also consistent with our assumption (Figure 4b). It could be concluded that 1.25 g/L of lipopeptide extract (containing 5.625μM fengycins) would be the optimal inhibitory concentration to be applied in our product.

![](https://static.igem.wiki/teams/4274/wiki/figure-4j.jpg)

Figure 4. Fengycins’ inhibitory effect on the quorum sensing system of Staphylococcus haemolyticus. (a) Biofilm quantitative detection of Staphylococcus hemolyticus at different concentrations of lipopeptide extract. (b) Measurement of the growth curve of Staphylococcus hemolyticus at different concentrations of lipopeptide extract.

# Target 3 Santalene production in E.coli DH5α

As stated in the project description, after engineering, E. coli could utilize both MEP pathway and MVA pathway for the universal precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), then synthesize santalene with the help of FPP Synthase (FPPS) and santalene synthase (SS). Except heterologously expressed MVA pathway and ERG20 of Saccharomyces cerevisiae and santalene synthase of Clausena lansium (ClSS), several modifications upon ERG20 or ClSS by amino acid mutation, binding to a hydrophillic tag and the construction of fusion protein were tested for the higher yield of santalene. Therefore, with the help of the co-transformation of pMVA plasmid with various pW1 plasmids, including pW1\_CE, pW1\_CEM, pW1\_TCEM and pW1\_CEM\_FL, different strains like CE, CEM, TCEM, CEM\_FL were successfully constructed (Figure 5). The complete pathway we designed for producing santalene in E. coli is illustrated in Figure 5.

![](https://static.igem.wiki/teams/4274/wiki/figure-5.jpg)

Figure 5. Construction and expression of santalene. (a) Enzymes and some of the reaction intermediates necessary for the production of santalene through the MEP pathway and MVA pathway. (b) Schematic representing the structure of pMVA, pW1\_CE, pW1\_CEM, pW1\_TCEM and pW1\_CEM\_FL transformed into E.coli DH5α ∆TnaA.

Afterwards, the various engineering of E.coli DH5α ∆TnaA mentioned above were used for santalene production. After rapid centrifugation, the supernatant of dodecane was spiked with with 0.475 g/L a-humulene as an internal standard, and then injected into GC/MS for verification of α-santalene production. It turned out that all samples from four strains appeared a significant peak at the retention time of 26-27 min, and various peak area of different samples exhibited santalene production with differing levels, indicating the general success of E. coli engineering. It can be concluded that the E. coli strain CEM (with pW1\_CEM plasmid) produces the maximal level of α-santalene compared to other strains (73.93 mg/L). Furthermore, our study elucidates that the mutation of 96th amino acid into tryptophan could increase the yield of α-santalene by about 20%, substantiating the prominent performance of ERG20F96W in enhancing the supply of FPP and α-santalene production in E. coli (Figure 6).

![](https://static.igem.wiki/teams/4274/wiki/engineering/figures/figure6.png)

Figure 6. Quantification analysis of α-santalene production. (a) Measurement santalene production of different strains by GC/MS results. (b) Quantification of α-santalene is analyzed with 0.475 g/L α-humulene as an internal standard. And the GC/MS results demonstrate that the peaks at the retention time of 26-27 min and 28-29 min respectively were α-santalene and α-humulene. (c) GC/MS results of samples originated from CE, CEM, TCEM and CEM-FL strains.

# Conclusion and Discussion

Our team had successfully engineered Bacillus subtilis 168 to produce fengycins, capable of inhibiting staphylococcal population, and biosynthesized α-santalene from E.coli, promoting a synthetic fengycins- based deodorant with the scent of sandalwood was developed.

Due to related policy that impose the ban of living bacteria application on exposed human body, part of our project cannot be implemented fully. If the limitation of biological synthesized product alleviate, we would proceed with the engineering of live bacteria hydrogel products that modify Bacillus subtillis by using its dtpt in the absorption of Cys-Gly-3M3SH on skin, the precursor of 3M3SH odor molecules. Our team would also introduce the suicide mechanism to ensure the safety and enhance deodorant effect, meanwhile avoid the addition of environmental burden.

Currently, our team succeed in knocking out the patB enzyme in the Bacillus subtilis 168, guarantee that Bacillus subtilis 168 can both absorb Cys-Gly-3M3SH and avoid the production of odor. This result had laid the experimental foundation and pave the way for our second-generation product (Figure 7).

The possibilities opened up by the AROMATA are endless. As people with body odor is faced with a rather severe social dilemma and in lack of a safer, more eco-friendly and more convenient solution. The AROMATA hopes to be a better solution to body odor, and become a new trend in the cosmetic industry.

![](https://static.igem.wiki/teams/4274/wiki/engineering/figures/figure7.png)

Figure 7. Screening results of Bacillus subtilis strains with successful patB gene knock-out. (a) Electrophoresis results show that gene editing is successful. (b) The schematic representation of Bacillus subtilis genome before and after patB being knocked-out.

{% endblock %}