Brigette O’Neill and George Jingyuan Wang
Western Reserve Academy, Hudson, Ohio, USA
Reviewed on 8 May 2021; Accepted on 28 June 2021; Published on 25 October 2021
With help from the 2021 BioTreks Production Team.
Keywords: Scent, fragrance, heat shock promoter, music, concert, violin, friction, bowed string
Authors are listed in alphabetical order. Beth Pethel mentored the group. Please direct all correspondence to pethelb@wra.net.
Background
As one of human’s irreplaceable senses, the sense of olfactory is largely undervalued in most cultures despite providing huge psychological and physical benefits. For example, fragrances provide strong cues for detailed past memories (Chu & Downes, 2002). With this special trait, scent is proven to be effective in the form of aromatherapy when applied to patients with Dementia or Alzheimer’s Disease (Jimbo et al., 2009). In daily lives, scent has the potential of becoming the most popular source of entertainment, as it can directly influence people’s memories and therefore leave the audience with some memorable experiences.
In our society, the value of scent remains largely unexploited. Scent is often treated as a condiment instead of a focal point: when entering a high-end clothing store, we can often smell the perfume that serves to elevate our shopping experience. Before attending an important meeting, people will spray perfume onto themselves and brush their teeth to avoid bad breath. In either case, scent, though crucial, is not the main focus of any part of our lives. Intending to fill in this gap, our team developed the idea of the scented concert–a revolutionary form of entertainment that aims to fully exploit the potential of fragrances and combine that with music.
Further investigation shows us that the combination of auditory and olfactory senses is particularly favorable for entertainment. Experiments by Professor Seo and Hummel (2011) from the University of Dresden Medical School have shown that there’s a clear link between the pleasure one felt toward an odor and the extent to which one enjoyed the preceding sound. As for our scented concert, this result indicates that the pleasure the audience will take from the fragrances will be boosted by the music they hear, resulting in a magnified multisensory experience that no current entertainment provides.
Another possible benefit of the scented concert is that it might be able to induce artificial synesthesia between sounds and scents. Researchers have shown that in general, people with synesthesia achieved better memory scores than the control group, especially in memories related to their connected senses (Tierney, 2019). Thus, musicians and perfumers may find the scented concert especially beneficial to their careers, so as the general public who might benefit from the improved memory.
To implement our idea, we decided to utilize the power of synthetic biology. Many pieces of research and experiments focusing on the use of biotechnology in the fragrance industry have been conducted by scholars. In a review written by several researchers from the Macquarie University, the authors claimed that “as already shown with the development of a hoppy yeast, many of the strategies employed and discussed [in the review] could easily be used to engineer aroma development in strains involved in the fermented beverage industry” (Wyk et al., 2018). The use of genetically engineered yeast to generate scents in the wine industry is just one example of many real-world applications of synthetic biology in the production of fragrances. According to Lux Research, “accelerating advances in our understanding of synthetic biology can unlock novel methods to produce flavor and fragrance chemicals that avoid [the drawbacks of conventional methods] altogether” (Lux Research, 2015). The drawbacks, as Lux claimed, are the “suffer from high cost, low sustainability, and high risk of supply chain disruption.”
In fact, several scented concerts have been held before by different artists. However, none of them attempted to set up a repeatable standard for this new artistic format. In 2004, an instrument known as “Olfactiano” (shown in Figure 1), designed by Belgian artist Peter de Cupere, was for the first time being played in a scent sonata. The instrument can release scents corresponding to the keys–similar to the ones on a regular piano–that are being pressed. Although the concert was successfully held, it was too costly and technically difficult to be copied in other places.
Nonetheless, synthetic biology provides a less costly and more sustainable alternative to realize our idea. We can reach the desired effect by transforming fragrance-producing genes into well-researched organisms such as Escherichia coli, which will be programmed to release fragrances as the music starts to play. Since E. coli can undergo binary fission and easily multiply in size over a few hours, scented concerts can be performed simultaneously across the world with little cost being added per show.
With these clear advantages, we are convinced that synthetic biology will provide the best solution to achieve our goal. The following sections of our paper will discuss the specific design we have from the perspective of system, device, and parts.
Systems Level
The system of our project consists of the natural heat shock that exists in most concerts and genetically edited E. coli that will be sprayed onto string instruments right before each performance. In the article, “Bowed String Simulation Using a Thermal Friction Model”, Woodhouse from Cambridge University analyzed the heat generated when a bow moves across strings (Woodhouse, 2003). His simulation result shown in Figure 2 suggested that when a string is bowed, therefore releasing heat into the surrounding space, the temperature above ambient varies between 17 and 31 degrees Celsius (Woodhouse, 2003). This shift in temperature indicated the presence of heat shock when string instruments such as violin are played by bows.
Taking advantage of the natural heat shock generated by string instruments during a concert, our system then includes synthetically engineered E. coli which can recognize the heat shock and activate the fragrance-producing genes contained in their transformed plasmids. The design will make sure that the production of fragrance and the performance of music will be generally in sync. Only when the E. coli are sprayed onto the string instruments and heat is produced from the bowed strings could the fragrance-producing genes be transcribed. The process is indicated in Figure 3. To ensure a high survival rate of E. coli when they depart from their cultures during the performances, they should be sprayed onto the strings immediately before each concert. Also, for the system to function properly, performers have to make sure that there isn’t any significant temperature difference between the E. coli cultures and the performance site.
Device Level
Plasmids containing four distinct fragrance-producing genes (FPGs) will be transformed into E. coli separately. We wish to match each string on a violin with a distinct aroma, therefore associating musical notes with specific scents. We identified four genes on the iGEM registry encoding for beta-Pinene synthase (BBa_M50438), MpAAT1 enzyme (BBa_K395600), Geraniol (BBa_K727007), and Santalene Synthase (BBa_K1322231), which can produce the scent of pine, apple, rose, and sandalwood respectively. As discussed earlier, we will insert a heat-shock inducible promoter known as the grpE promoter (BBa_K338001) before the FPGs so that the scents can be released in sync with the music. The promoter, like other heat-shock inducible promoters in E. coli, regulates the σ-32 factor. σ-32 controls heat shock response by directing RNA polymerase to transcribe heat shock proteins, which are synthesized when the temperature rises (Nonaka et al., 2006). To increase the efficiency and accuracy of the translation of the genes, a strong ribosome binding site (BBa_B0030) will be placed in front of the FPGs. Finally, the double terminator rrnBT1-T7TE will be assembled after the FPGs. This terminator is the most commonly used terminator according to the iGEM registry and seems to be reliable (Shetty, 2003). Due to the lack of plasmids containing the aforementioned parts, we decided to synthesize them manually with their complete sequences that can be found on the iGEM registry. Since E. coli is one of the most studied bacteria and is commonly used in synthetic biology, it will be an ideal chassis for the purpose of our project. All of our parts are either designed to be transformed into E. coli or proven to be expressed in E. coli (Matsubara, 2010; Orhan, 2012; Schreiner, 2018; Shilts, 2014) . The way in which our FPGs produce fragrances is summarized in Figure 4.
The first FPG, shown in Figure 4.1, codes for beta-Pinene synthase, an enzyme that converts GPP (geranyl diphosphate) into beta-Pinene (Schreiner, 2018). Beta-Pinene is a molecule that produces pine fragrance. In E. coli, Mevalonate (MVA) and methylerythritol phosphate (MEP) pathway naturally produce GPP from dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP), allowing the device to function (Zhou et al., 2015). Figure 4.2 demonstrates the mechanism of the apple FPG, which codes for the enzyme MpAAT1, which catalyzes the production of Butyl acetate and 2-Methylbutyl acetate from butanol and 2-Methyl-1-butanol (Matsubara, 2010). Both Butyl acetate and 2-Methylbutyl acetate can release apple scents. Acetyl CoA, the substrate of MpAAT1 enzyme, naturally exists in E. coli cells. Thus, in order to produce apple fragrance, butanol and 2-methyl-1-butanol should be applied to the E.coli culture containing the transformed apple FPG before performances. The third FPG, depicted in Figure 4.3, includes genes that code for GPP synthase and geraniol synthase (Orhan, 2012). GPP synthase will catalyze the production of GPP from IPP and DMAPP as discussed above, and geraniol synthase will produce geraniol from GPP, which is a chemical that has a rose fragrance (Orhan, 2012). The sandalwood FPG, shown in Figure 4.4, translates into alpha-Santalene synthase, an enzyme that converts Farnesyl pyrophosphate (FPP) into (+)-alpha-Santalene (Shilts, 2014). FPP is produced in E. coli as an intermediate in several pathways, including MVA and MEP (Shilts, 2014). (+)-alpha-Santalene, on the other hand, is a sesquiterpene (a kind of compound) that releases sandalwood scents.
Parts Level
Following the procedures depicted in Figure 5, we will assemble the parts mentioned above into the vector backbone pL1V-Lv1-ColE1-amp, a high-copy empty backbone used for E. coli transformation (Geddes et al., 2019). All FPGs except for the rose FPG will be cloned into the plasmid through the XbaI restriction site. The rose FPG, on the other hand, will be cloned using the XmaI restriction enzyme due to the existence of XbaI restriction sites within the gene. As both XbaI and XmaI restriction sites are located inside the LacZ gene within the plasmid, the correctly transformed plasmid will contain disrupted LacZ genes. Thus, after the transformation, blue-white screening can be applied to identify the successfully transformed E. coli, which will appear white under screening.
Safety
Generally, there should not be any safety concerns with our proposed experimental design. We will be working with the DH5 alpha strain, which is a non-pathogenic E. coli strain. All procedures will be conducted in a Biosafety Level 1 laboratory (BSL-1) environment. However, we recognize the possibility of physical contact and inhalation of E. coli cells due to their complete exposure during the performance. Although little or no health issues should result from this, we recommend all performers and audiences who sit close to the stage to wear masks, gloves, and eye goggles during the performance. All participants, both the musicians and the audience, should wash their hands with soap thoroughly after the concert. Meanwhile, the instruments being sprayed with E. coli cells should be cleaned thoroughly following appropriate procedures.
Discussions
Although the design should theoretically work, several problems might impede us from achieving the desired result. First and foremost, there have not been any previous experiences with the four fragrance-producing genes that can prove their effectiveness, nor sufficient records of their usages on the iGEM registry. Compared to the FPGs, all other parts including the heat shock promoter, the RBS, and the double terminator are much more frequently used and reliable.
A second major issue is whether the amount of heat shock generated by the friction between violin bow and strings is enough to activate the grpE promoter, and whether the amount of fragrance produced is able to disseminate through the entire concert hall. An experiment with the grpE promoter fused to Green Fluorescent Protein (GFP) revealed that after applying heat shock to E. coli cells at 42°C for 0 (control), 5, and 10 minutes respectively, only the group that received 10 minutes of continuous heat shock gained a significant increase in GFP production, while the 5-minute group showed little difference from the control group (2010 Caltech iGEM Team, 2010). Nevertheless, another experiment with the gprE promoter suggested an increase in transcriptions activated by the heat shock promoter as temperature increases (2010 Caltech iGEM Team, 2010).
The experiments cast suspicion on the assumption that the heat generated by the bowed strings during the concert can immediately lead to a noticeable amount of fragrance production. Further research and experiments are required to determine if there will be a significant time lag between the onset of music and the production of scents and whether the temporary pauses of heat production during the performances would have significant effects on the expression of FPGs. On-site investigations will be conducted to measure the strength and distinctiveness of the fragrances produced and how well they spread across the room. .
Another concern is the survival rate of the E.coli cells without the support of nutrients and an optimal temperature from their cultures during the performances. According to a study conducted by the Spanish Ministry of Science and Innovation, after leaving two groups of E.coli cells in nutrient-deprived environments for 21 days at 4 ℃ and 20 ℃ respectively, the absence of nutrients had no effect on cell viability and integrity, though their culturability declined faster at a higher temperature (Orruño et al., 2017). Nevertheless, at the end of 3 days, almost all cells (>90%) experimented at both high and low temperature levels remained both culturable and viable (Orruño et al., 2017). Hence, we are confident that our E. coli will be able to persist during a scented concert even without the support of their cultures.
Besides the practicality of our idea, we also identified some potential defects with our specific design. First, it is unclear whether the act of spraying E. coli onto the violin strings can cause damage to the strings, the bow, or the violin itself. According to Marshall (2015) , excess humidity can affect the texture of the bow and thus hurt its performance during the concert. Therefore, it is reasonable to believe that the E. coli cells, sprayed onto the strings as liquid solutions, will interfere with the musical quality of the instruments. In the long term, the enduring existence of moisture on the instruments might also shorten their longevity. However, we will need to analyze the exact side effects of the presence of E.coli on the strings by comparing the instruments used in the scented concerts with the ones used in regular concerts. If severe side effects are identified, we will develop other methods that enable our device to work properly on the strings while causing no or minor damage to the instruments.
Another problem is that our chassis, E. coli, is notorious for its bad smell due to the reaction of the tryptophanase enzyme that produces indole, a smelly chemical (Remove the Bad Smell, 2014). The bright side is that several teams have already developed mechanisms to eliminate their bad smells (Remove the Bad Smell, 2014). In addition, this is not a serious setback considering all the advantages of working with E. coli–an accessible, safe, and well-known organism.
Next Steps
The next step of our project is to synthesize such a device and test it in a controlled environmental setting. This might be the best way to find solutions to the concerns stated in the previous section. We also encourage other individuals and groups to develop further research and experiments based on our model. Bearing all the challenges and concerns in mind, we still believe that our project contributes an innovative idea and approach to create an avant-garde. By further modifying our design and testing it with experiments simultaneously, if we do eventually conquer the aforementioned challenges, our scented concert will transform the musical experience for all listeners, providing an unprecedented multisensory enjoyment.
Author Contributions
B.O. assisted in the preliminary research for this project. G.W. contributed the original idea and identified all necessary parts for the device. G.W. also did all the writings and research for all sections of the paper.
Acknowledgements
We would like to acknowledge our institution, Western Reserve Academy, for providing us the necessary resources to complete our project. I(G.W.) would especially like to thank Dr.Pethel for guiding me meticulously through my first and last major biology project in high school.
References
Chu, S., & Downes, J. J. (2002). Proust nose best: Odors are better cues of autobiographical memory. Memory & Cognition, 30(4), 511-518. https://doi.org/10.3758/BF03194952
Frank, P. (2014, October 23). Olfactory artist has crafted a ‘Holy Water’ sculpture that smells like vagina. HuffPost. https://www.huffpost.com/entry/vagina-smell-sculpture-_n_6031876?slideshow=true
Geddes, B. A., Mendoza-Suárez, M. A., & Poole, P. S. (2019). A bacterial expression vector archive (BEVA) for flexible modular assembly of golden gate-compatible vectors. Frontiers in Microbiology, 9, 3345. https://doi.org/10.3389/fmicb.2018.03345
Seo, H. S., & Hummel, T. (2011). Auditory–olfactory integration: Congruent or pleasant sounds amplify odor pleasantness. Chemical Senses, 36(3), 301-309. https://doi.org/10.1093/chemse/bjq129
Jimbo, D., Kimura, Y., Taniguchi, M., Inoue, M., & Urakami, K. (2009). Effect of aromatherapy on patients with Alzheimer’s disease. Psychogeriatrics, 9(4), 173-179. https://doi.org/10.1111/j.1479-8301.2009.00299.x
Lux Research. (2015, July 20). Eau d’coli: Application of biotechnology in flavors and fragrances. Lux Research. https://members.luxresearchinc.com/research/report/17318
Nonaka, G., Blankschien, M., Herman, C., Gross, C. A., & Rhodius, V. A. (2006). Regulon and promoter analysis of the E. coli heat-shock factor, σ32, reveals a multifaceted cellular response to heat stress. Genes & Development, 20(13), 1776-1789. https://doi.org/10.1101/gad.1428206
Marshall, E. (2015, March 5). A matter of balance: How to manage the humidity level of your instrument. Strings. https://stringsmagazine.com/a-matter-of-balance/
Matsubara, T. (2010, October 4). Registry of standard biological parts: Apple fragrance enzyme (MpAAT1) generator. iGEM http://parts.igem.org/Part:BBa_K395600
Orruño, M., Parada, C., Kaberdin, V. R., & Arana, I. (2017). Survival of Escherichia coli under nutrient-deprived conditions: Effect on cell envelope subproteome. Escherichia coli-Recent Advances on Physiology, Pathogenesis and Biotechnological Applications. http://dx.doi.org/10.5772/67777
Orhan, I. Y. (2012, June 16). Registry of standard biological parts: Rose odor generator device (Geraniol). iGEM. http://parts.igem.org/Part:BBa_K727007
Remove the bad smell of E.coli. (2014). http://2014.igem.org/Team:Paris_Saclay/Project/
Schreiner, G. (2018, April 25). Registry of standard biological parts: Beta pinene synthase sequence. iGEM. http://parts.igem.org/Part:BBa_M50438
Shilts, J. (2014, October 16). Registry of standard biological parts: Santalene synthase. iGEM. http://parts.igem.org/Part:BBa_K1322231
Shetty, R. (2003, July 17). Registry of standard biological parts: Double terminator (B0010-B0012). iGEM. http://parts.igem.org/Part:BBa_B0015
Tierney, W. (2019, May 14). Ask a biologist: Is The Letter “A” Red?. Arizona State University. https://askabiologist.asu.edu/plosable/letter-red
Van Wyk, N., Kroukamp, H., & Pretorius, I. S. (2018). The smell of synthetic biology: Engineering strategies for aroma compound production in yeast. Fermentation, 4(3), 54. https://doi.org/10.3390/fermentation4030054
Woodhouse, J. (2003). Bowed string simulation using a thermal friction model. Acta Acustica United with Acustica, 89(2), 355-368.
Zhou, J., Wang, C., Yang, L., Choi, E. S., & Kim, S. W. (2015). Geranyl diphosphate synthase: An important regulation point in balancing a recombinant monoterpene pathway in Escherichia coli. Enzyme and Microbial Technology, 68, 50-55. https://doi.org/10.1016/j.enzmictec.2014.10.005
2010 Caltech iGEM Team. (2010, October 24). Registry of standard biological parts: Heat shock promoter (HSP). iGEM. http://parts.igem.org/Part:BBa_K338001