KS&DL Banglore
Karnataka Sandalwood Soaps and Detergents Limited, Bangalore. A government enterprise producing sandalwood products, home to the iconic Mysore Sandalwood Soap of India.

During our visit to KS&DL, our goal was to learn more about the production and demand for sandalwood oil products, particularly the Mysore Sandal Soap, so we talked to Mr. Manoj. We inquired about the recent article that inspired us, and he confirmed the unfortunate situation: this year has been challenging, and the company had to import from Australia. There's also uncertainty about the availability of sandalwood oil in the coming years. This shortage poses a significant issue not only in Indian demand but also in exports.

KSDL is known for a myriad of products that use various essential oils such as jasmine, neem, etc, which are more flexible in their supply compared to sandalwood oil. We learned that the demand for these essential oils could always be met with production. However, that was not the case for sandalwood oil owing to its resource-intensive methods of production.

“We envision a promising future for sandalwood soap, but it just isn't possible without the authentic Indian sandalwood oil.”

Mr Manoj was quite impressed with PETal and our novel approach to producing sandalwood oil. Additionally, they pointed out how an additional source of sandalwood oil would be highly beneficial in addressing the growing market demands. However, they raised concerns about its authenticity, specifically on how our product's composition would differ from the one naturally derived from Indian sandalwood trees.

Holy Lama Naturals
Mr Vijay Deo from Holy Lama Naturals. Kochi-based company produces cosmetic products using essential oils. Market presence in the UK, Netherlands and the Middle East.

He gave us a tour of their operations. Holy Lama offers a range of around 200 sandalwood products, including soaps, incense, ubtan, and aromatherapy essentials. However, with the escalating cost of Indian sandalwood oil, even private companies like Holy Lama are struggling to afford it. As a result, they have recently started sourcing sandalwood oil from Givaudan, a Swiss-based global leader in fragrances and beauty. However, this oil is made from Australian sandalwood trees.

While Holy Lama is committed to sustainability and already uses eco-friendly packaging, a more affordable and sustainable alternative to sandalwood oil would greatly support their efforts.

Reflections:

Recognising the high demand, we wanted to ensure our product could scale to meet current needs without compromising environmental sustainability. Taking a bench-to-bedside approach, we explored the industrial applications of our project. This also prompted us to explore future directions for PETal beyond just producing from PET. With PETal, we can produce various components of sandalwood oil, formulate them into different compositions, and incorporate them into a variety of beauty and cosmetic products.

Officer Sanoj S
Range Forest Officer, Aryankaavu Sandalwood Protection Reserve, Kollam, Kerala

To further investigate the sandalwood shortage, we visited Aryankaav, a sandalwood protection reserve near our campus. Range Forest Officers provided valuable insights into the state of sandalwood cultivation and protection measures.

They explained the significant space requirements for sandalwood tree plantation, noting that a single tree, along with its hemiparasitic plants, can require up to 6 square meters and take over 30 years to mature. Tropical climates are ideal for growth, restricting it to southern India and Australia, and propagation typically occurs through roots as well.

The officer highlighted the challenges of theft and animal conflicts, as young sandalwood trees are particularly vulnerable due to their weak trunks. Smuggling is a frequent issue, given the high value of the wood. To give us a firsthand understanding, we were taken on a jeep tour through the reserve, where we observed the extent of damage caused by even minor animal disturbances or smuggling. Here, we also had the opportunity to meet the Vanasena, the protectors of the sandalwood trees running around the clock for patrolling, whose efforts we later showcased in an "Unsung Hereos" documentary.

Due to these threats, the Government of India refined the imposed strict regulations under the Sandalwood Protection and Regulation Bill of 2008. Which states that cutting sandalwood trees requires authorisation from the Forest Department, and individuals are restricted from possessing more than 1 kg of sandalwood. Any excess must be transported to Marayoor, the central hub for government-regulated sales.

This further made us talk to Marayoor Sandalwood division.


Officer Abju K Arun
District Officer Head of Marayoor Sandalwood Reserve, Kerala’s largest Sandalwood Reserve Protection.

This reserve oversees a 65-sq-km home to approximately 57,000 sandalwood trees. During an interaction with the DFO (District Forest Officer), he shared insights into the extensive measures undertaken to protect this valuable species. These include camera surveillance, geo-tagging, 24/7 monitoring, and perimeter fencing, all funded by the government to safeguard India’s Royal tree.

“These measures are essential, as incidents of smuggling or animal conflicts that take place weekly threaten years of conservation efforts,” said Arun.

Similar to the Aryankaavuu, this place in the past, the reserve faced a significant decline in sandalwood trees due to rampant smuggling and animal conflicts. Arun emphasised that despite the government's efforts, the demand continues to outpace supply, with only 550 kg of sandalwood being auctioned daily. Marayoor remains the sole auction port for all sandalwood reserves, underscoring the challenges of managing this high-demand yet low-supply resource.

Reflections:
Our interactions with the Forest officers further cemented the need for PETal. Our visit to the largest sandalwood reserves in both India and Kerala was pivotal in gaining insights into the importance of sandalwood cultivation and the need for protection measures and sustainable practices. Gaining a first-hand look into the current extensive efforts taken by the forest department in India, we understood that the current methods are extremely costly, resource and labor-intensive in meeting the growing market demands in the long run. The conservation efforts of Indian sandalwood trees are crucial because this species is threatened^[1], and over-exploitation has led to the decline of this natural gem. PETal aims to alleviate pressure on the existing sandalwood supply by introducing an alternative source of sandalwood oil.

Abdul Vahid

SS Scrape Business, a Private Plastic Processing Unit near our campus in Vithura (Kerala Pollution Control Board approved)

We found inspiration and knowledge every single day of working on PETal. One sunny morning, on our way to the waterfalls, we noticed a large plastic processing unit and decided to inquire about the private sector of plastic waste management. Abdul Vahid, the owner of this business, shared that their facility processes 8 tonnes of plastic daily, including 1 tonne of PET collected from Vithura, Nedumangad, and Palode in Kerala. Their processes include human-based segregation, machine chipping, and compression for compact packing and further transport.

He explained that PET can be recycled only once effectively, losing its properties in subsequent rounds. Their approach involves upcycling PET by sending it to a Gujarat-based company, Dulex Recycling. This company converts the compressed PET into nylon threads and t-shirts. This provides a good amount of incentives for better plastic management for the general public as opposed to initiatives taken by the government municipality.

“PET is unavoidable; the majority of it is made of crude petroleum, and recycling it still keeps it in the environment as petrochemicals.”

Reflections:

Living in a society where the use of plastic is so widespread and normalised, active efforts to change the system are difficult but necessary. Small steps taken by these private plastic management companies, such as giving more incentives to the public to recycle their waste, can slowly help in the shift towards sustainable living. In this regard, we took the beach cleanup initiative; the link is here. We also realised the importance of plastic management awareness amongst the public, inspiring our educational activities further.

Ambika Vasudev

Private Sandalwood Cultivator in Hassan, Karnataka.

During a wedding attended by our team, we had the opportunity to speak with Ambika Vasudev, a private sandalwood cultivator, in hopes of gaining insights into private sandalwood cultivation. Ambika has experience of 6 years, and her plantation covers 5 acres of land.

Ambika highlighted the significant gap between the procurement prices offered by government agencies and the retail prices through which sandalwood is sold. This discrepancy serves as a disincentive for sandalwood farmers. Cultivating sandalwood is no small feat, requiring a considerable investment of time and resources. The trees take around 30 years to mature, and the risks associated with them are high. Moreover, the cultivation process is challenging due to the fear of smuggling and the lack of insurance schemes, so any case of robbery will fall straight on the owner. Sandalwood trees also require being planted with other species as they are hemiparasites.

“It’s a long-time investment, with lots of costs and risks.”

Reflections:

Even private cultivators face significant threats to sandalwood production, and if they manage to mitigate these risks, it often comes at a considerable cost. In contrast, the government reserves benefit from state-funded protection and dedicated officials, yet conditions remain challenging, underscoring the urgent need for innovation.

We incorporated surveys to gather input from the largest group of stakeholders. Surveys are an effective data collection tool that allows researchers to gain insights from diverse populations and identify general trends or common solutions. However, like any research method, surveys require careful planning and design to ensure accuracy. A concise and well-structured survey featuring unbiased wording, clear response categories, and straightforward questions can yield valuable results.

Dr Harilal Madhavan from our institute, introduced us to the concept of human subject research and emphasised the importance of adhering to its guidelines throughout our events. Since then, we have consistently followed the necessary protocols to respect the interests of our participants. Throughout our project, our team organised numerous online and offline meetings, webinars, surveys, interviews, camps, and competitions to raise awareness and gather valuable insights for improvement. We ensured full compliance with all relevant laws, regulations, and institutional guidelines pertaining to Human Subject Research.

Consumer Survey
The survey aimed to assess the general public, who are our end consumers, and their perceptions of synthetic biology products while exploring concerns about GMO-based (Genetically Modified Organisms) products and sustainability awareness. It also examined how views differed by age and profession and analysed consumer behaviour to identify target segments.

Most respondents in our survey demonstrated a solid understanding of synthetic biology and its applications, particularly regarding GMOs in product production. While many expressed a neutral to positive stance towards products derived from synthetic biology, concerns about consumer safety 25.2% and environmental impact 23.2% emerged as the primary apprehensions. Additional worries included product efficacy 18.6%, ethics 15.1%, and cost 14.8%.

Notably, nearly 60% of participants emphasised the importance of sustainability in their purchasing decisions. The survey also revealed insights into consumer preferences for sustainable products: 69% favoured affordable, sustainable options, while 24% were willing to pay a premium for such products. Despite the concerns, a significant majority indicated a strong willingness to consider products made through synthetic biology. This positive response highlights the potential for innovative solutions that address both consumer desires and environmental challenges, paving the way for more sustainable practices in the beauty industry. Demographics with respect to age was biased towards 18-25 years, and mostly being students.

Reflections:
We realised that the general public is willing to adopt sustainable products. However, there are still certain concerns associated with the usage of GMOs, among which environmental impact and safety of the consumer are the ones that were voted for the most. It's promising to see society recognise environmental degradation and take individual actions to help reduce its impact. We gained insight into how much the respondents were aware of and how much extra they would be willing to pay. This gives us a scope to do a cost analysis, take a look at it in detail on the Entrepreneurship page.

Synbio survey
We conducted surveys aimed at understanding the gap between scientific knowledge and the community before launching our education activities, allowing us to tailor our outreach efforts accordingly. The majority of participants were students, and the surveys covered key concepts in synthetic biology.

Reflections:
Through these survey, participants rated their understanding of important synthetic biology terms and provided feedback on topics they wanted to learn more about. This valuable input helped us identify areas where scientific communication could be improved and develop an audience-centric approach. This catered to the designing of the outreach on the Education page.

Dr Joanna C Sadler

Joanna Sadler is a Chancellor's Fellow in Biotechnology at the University of Edinburgh, specialising in innovative approaches to plastic waste management. She is recognised for her groundbreaking work in upcycling PET plastic into vanillin using engineered E. coli, marking a significant advancement in sustainable biotechnology and addressing environmental challenges related to plastic pollution.

• Reasons to Approach: Since the whole project was inspired by Joanna C. Sadler's groundbreaking research on the same, it was necessary to consult her for designing our project, exploring the feasibility of PET as an ideal feedstock for the production of our sandalwood oil.


• Suggestions: Her inspiration came from the pressing issues of plastic pollution, the need for a circular economy, and the demand for vanillin. She mentioned that working with metabolic pathways involving terpenoids is relatively easy. She suggested the use of LC cutinase to break down PET into monomers, as it directly releases TPA without the need for an additional enzyme like MHETase. She also recommended incorporating Flux Balance Analysis (FBA), which she couldn't implement herself but believes is crucial, based on advice from the expert she approached. While she used HPLC to detect the compound, she noted that better methods exist if accessible. Additionally, she suggested we consider the importance of in-situ product removal (ISPR) for our model and highlighted the importance of educating others in the industry that compounds produced through synthetic biology are chemically identical to their natural counterparts

• Integration: Based on her suggestions, we integrated FBA into our model to optimise its performance, which eventually led us to Dr Sanu from IISER TVM. We also sought out additional experts and employed GC-MS for the detection of santalene and santalol. For the proposed implementation phase, we began exploring the incorporation of LC cutinase for the depolymerisation process and took proactive steps to educate the public about synthetic biology products.

Dr Oliver Brandenberg

Dr Brandenberg is a researcher at the Swiss Federal Institute of Aquatic Science and Technology (EAWAG), specialising in plastic biodegradation, directed evolution, and green chemistry; his work includes engineering Pseudomonas putida to utilise terephthalic acid (TPA) and ethylene glycol (EG) as sole carbon sources.

• Reasons to Approach: We reached out to Dr Brandenberg due to his expertise in engineering microbial strains for specific metabolic pathways and his experience with Pseudomonas putida. Given the complexity of our project, which involves engineering a strain to both take-up PET monomers and produce santalol, his insights were crucial.


• Suggestions: After reviewing our proposed timeline, he concluded that engineering a new organism from scratch alongside developing the santalol pathway was too ambitious within the given timeframe. Instead, he recommended leveraging his existing engineered strains of Pseudomonas putida TA7-EG ^[2]. He advised that we focus on remodelling the metabolic pathways already present in these strains to build up on our terpenoid production. Additionally, in the second iterative cycle, he suggested adjusting the growth conditions with M9 minimal media, preferably liquid culture, in this case, to grow the strains. He also emphasised the value of employing adaptive evolution techniques to enhance strain performance under specific conditions further.

Integration: As shown in the Engineering Page cycle 1, we decided to move forward with the integration of the already-engineered bacteria. We made the necessary adjustments to the media composition using adaptive evolution techniques. By gradually reducing glucose levels through serial dilution, starting from 5% and decreasing to 1%, the bacteria increasingly rely on TPA and EG, we enhanced their performance within the system.

Dr Jordi Perez

A Marie Curie Fellow at the Centre for Research in Agricultural Genomics (CRAG) in Barcelona, Spain, specialising in isoprenoid biosynthesis, synthetic biology, and metabolic engineering.

• Reasons to Approach: As an expert in isoprenoid synthesis, we reached out to Dr Jordi Perez for his guidance on optimising the metabolic engineering of our pathway. Specifically, we were exploring suitable pathways for the production of santalol and its precursors, DMAPP and IPP, in our chosen chassis.


• Suggestions: After exploring pathways suitable for our chassis, Dr Perez recommended the MEP pathway for producing DMAPP and IPP, as it is native to Pseudomonas putida and offers greater efficiency compared to the MVA pathway. The MEP pathway requires fewer carbon atoms from pyruvate and G3P to produce the same amount of IPP and DMAPP while also placing less strain on acetyl-CoA, which is crucial for other metabolic processes.
  Additionally, he confirmed through experimental evidence that the MEP pathway is superior.
  We also learned that overexpressing DXS/DXR would enhance the abundance of santalene in our production process^[3].

Integration: Based on Dr Perez's guidance and our experimental findings, we opted to utilise the MEP pathway for our system. We incorporated DXS/DXR enzymes to enhance the production of santalene, for which he provided the necessary DXS/DXR plasmids, as depicted in cycle 4 of Engineering page.

Prof. Jörg Bohlmann

Prof. Bohlmann is a researcher at the Michael Smith Laboratories, University of British Columbia, Vancouver, Canada, specialising in terpenoid bioproducts and Cytochrome P450 enzymes.

• Reasons to Approach: We consulted Prof. Bohlmann due to his expertise in terpenoids and Cytochrome P450 enzymes, which are crucial for our project involving the production of santalene and santalol. His insights were sought to overcome challenges in the expression of santalol in our chosen chassis, as he previously worked with E. coli and S. cerevisiae and expressed santalene and santalol, respectively.


• Suggestions: He recommended utilising the FPPS_SaSSY_pCDFDuet vector system, which has been successfully employed in E. coli ^[4], to facilitate the production of santalene, a precursor to santalol. Although on the other side, he expressed concerns about the difficulties of expressing Cytochrome P450, required for the production of santalol from santalene in Pseudomonas putida, noting that it is a membrane-bound enzyme and our chassis is a prokaryote that may struggle with the necessary post-translational modifications. He highlighted that this could make the process of expression of santalol more intensive and challenging.
  

Integration:Following his recommendations, we integrated the pCDFDuet vector system into our chassis. For the cytochrome P450, we adopted strategies to engineer and express eukaryotic plant P450 in a prokaryotic system and began exploring other comparable systems naturally found in P. putida, refer to the Design page for more information.

Prof. Víctor de Lorenzo

Professor Lorenzo is a researcher at the Centro Nacional de Biotecnología-CSIC in Spain, specialising in synthetic biology, environmental biology, and metabolic engineering.

• Reasons to Approach: We contacted Prof. Lorenzo for his expertise in synthetic biology and metabolic engineering in P. putida, particularly for his insights into vector systems and choice of plasmid to design bicistronic, two genes under the same promoter system and the optimisation of gene expression for our chassis.


• Suggestions: After thorough discussions and troubleshooting regarding the functionality of the pCDFDuet vector, Prof. de Lorenzo advised that we develop our own constructs using the pSEVA vector system due to uncertainties surrounding successful expression. The pSEVA system is designed with a modular architecture that allows for easy assembly and customisation of genetic constructs, making it suitable for various applications. He provided critical guidance on promoter selection, spacer sequence length, and ribosome binding sites (RBS), all of which are essential for optimising gene expression. Prof. de Lorenzo validated our genetic insert designs, instilling confidence in the potential of our system and refining our approach, confirming the viability of our constructs.
  

Integration: To make our own constructs, we utilised the pSEVA241, pSEVA631, and pSEVA424 plasmids backbones from the pSEVA collection. A key integration involved adding an extended Shine-Dalgarno (SD) sequence adjacent to the stop codon of the preceding gene to enhance translation efficiency. Here is Design page that clarifies the above.

Dr Vijay Jayaraman

Dr Vijay is a researcher at the Indian Institute of Science Education and Research (IISER) in Thiruvananthapuram, India, specialising in enzyme engineering, synthetic biology and systems biochemistry. He also serves as the primary PI for our team.

• Reasons to Approach: We sought Dr Vijay's expertise due to his extensive synthetic biology experience and role as our primary PI. His involvement was crucial throughout our iGEM project, providing guidance in wet lab as well as dry lab aspects and administrative tasks related to iGEM permissions.


• Suggestions:Dr Jayaraman assisted us from the very beginning of our project, contributing to the ideation phase and helping with plasmid design strategies. He offered critical advice on choosing suitable promoters, such as araBAD and rhaBAD. He also validated our concept of a CPR-P450 fusion protein. Dr Jayaraman’s PhD student, Aswathy, provided invaluable support in helping the wet lab team with Gibson assemblies and electroporations. He dealt with planning experiments for growth curves by analysing the data. Dr Jayaraman’s proactive involvement and expert input were instrumental in refining our experimental strategies and ensuring the success of our project.
  

• Integration: All over our project, we integrated the suggestion given by him in wet lab and dry lab, especially in plasmid designing for selection of promoter, linker for fusion protein and overhang sequences of Gibson fragments were of utmost importance linked here. We consistently employed the Gibson assembly protocol he provided.
  

Prof. Shekhar Mande

Professor Mande currently holds a Distinguished Professorship at Savitribai Phule Pune University and is the former Director General of the Council of Scientific and Industrial Research. His expertise lies in the realm of structural and computational biology.

• Reasons to Approach: We were grateful for the opportunity to attend Prof. Mande’s talk at IISER TVM, which led to a productive discussion with him, the best of his kind in the field. His expertise in computational systems biology made him an invaluable resource for validating our project’s approach.


• Suggestions: Prof. Mande reviewed our wet lab and dry lab strategies up to the current stage and validated our overall approach till then. He assisted with the exploration and selection of different linkers to test the functionality of our fusion protein’s performance towards the end, suggesting GST linkers (Glycine-Serine-Threonine linkers), which would provide optimal functionality and flexibility for our constructs by preventing the formation of secondary structure form, which might hinder protein folding and function. His insights have been crucial for the fusion protein systems.
  

• Integration: His advice was the cherry on top. We proceeded with designing a modular GST linker part with flanking Sca I sites such that it could be replaced with any other linker he recommended and incorporated into our plasmid design with further details on modeling page.

Dr Sanu Shameer

He is a researcher at the Indian Institute of Science Education and Research (IISER) Thiruvananthapuram, specialising in systems and computational biology.

• Reasons to Approach: We approached Dr Shameer to assist with developing a Flux Balance Analysis (FBA) model, which was essential for monitoring inputs and outputs and optimising our pathways to predict the conversion efficiency of PET to sandalwood oil. His computational biology and systems modelling expertise made him a key resource for this task.


• Suggestions:Dr Shameer, along with his project student and our student advisor, Pratham, helped us address errors in our FBA model and provided foundational guidance on the basic concepts of flux balance analysis. He offered valuable insights that significantly improved the accuracy and functionality of our model, aiding us in making informed predictions about the conversion process and optimising our metabolic pathways. Together, we worked through troubleshooting to co-optimize the integration of santalol production and biomass as objective. His contributions were crucial for refining our computational approach and enhancing the overall performance of our model.
  

• Integration: After considering the above advice given and refining our model multiple times by (detail), We conducted a preliminary estimate to determine the potential yield of santalol from PET.
Check out the FBA section under modelling.

To understand if our transformed bacteria are expressing santalene when grown on TPA and EG, we needed to perform quantitative measurements using advanced techniques like GC-MS. We conducted these measurements at our campus’s CIF facility with the assistance of Dr Veera Reddy and his PhD student, Girish, along with KB Rameshkumar from JNTBGRI. As suggested by Joanna, we are also planning to perform further analysis using LC-HRMS to deepen our understanding of the results.