Human Practices

Introduction

Limonene and perillyl alcohol (PA) are valuable chemical compounds (monoterpenes) extracted from the essential oils found in certain citrus fruits (PA is also extracted from lavender). Limonene is widely used in foods (permitted by the USA Food and Drug Administration (FDA)), cosmetics and cleaning products due to its pleasant citrus smell (Bhatia et al., 2008)(Chen et al., 2015). Worth ~1.2 billion in the USA in 2018, the limonene industry was expected to increase by 5% by 2024 (Pulidindi, 2018). Perillyl alcohol is present in the food and cosmetics industries but is used significantly less than limonene (Chen et al., 2021). Despite this, PA in particular has shown very exciting clinical trial results where it is more potent than limonene (Crowell et al., 1994). Consumption of 2% PA over 10 weeks showed regression in both small and advanced carcinomas, not only this, but PA has also been shown to arrest any tumour cell growth (Haag & Gould, 1994)(Yuri et al., 2004). The emergence of intranasal administration, in which PA has been shown to cross the blood-brain barrier and increase the survival time of patients with recurrent glioblastoma, has reinvigorated the research into PA as an anticancer drug (Chen et al., 2021)(Da Fonseca et al., 2010). This, alongside potential chemoprotective effects on the skin, means that PA is a very valuable compound and one that we are passionate about increasing its production (Barthelman et al., 1998).

Our overall goal is to develop a perillyl alcohol (and limonene) production system in BL21 cells that can use certain encoded mechanisms to mediate the toxic effects of the products. If the toxic effects of the products are lowered, then it will result in more cells surviving that can produce more PA and a higher overall yield. The following sections will walk you through our values as a team and how we went through our project while maintaining them.

Team Diversity

At BioMonix we believe that having a diverse team would offer numerous perspectives and lots of varied strengths that mean we could tackle a complex problem from numerous angles. As such, including everyone, regardless of their academic, ethnic and socioeconomic backgrounds as well as ensuring a diverse selection of genders, ages and personalities was a very high priority. Making each team member feel comfortable to say what is on their mind and give their opinion on each step of the project is something that we aimed to maintain throughout our project. Offering flexible lab hours for those relying on part-time jobs and allocating space for one member's assistance dog just outside of the lab are just some of the actions we took to promote inclusivity. Find out more on our inclusivity page.

Education

Synthetic biology is an exciting field of science that is in a variety of fields (including therapeutics, agriculture and manufacturing just to name a few). Because of how applicable it is, we aimed to educate not only people involved in iGem or synthetic biology but also people who haven’t heard of iGem at all. One way we introduced the concept of synthetic biology to younger UK students was through in-person workshops delivered by members of our team at local schools. Getting the young people involved and thinking outside of the box to come up with their iGem ideas was very rewarding, more information can be found on the education page. Also along these lines, partnering with five other UK teams (hosted by King’s College London) we produced an educational package containing all the essential information needed for iGem (more info found in the education page). In an attempt to capture the interest of Warwick students from a variety of disciplines for upcoming years of iGem, we held a stall at the welcome fair and various welcome events (shown in the images below).

In an interest to see the different approaches teams had taken and what creative solutions they had come up with, we attended a UK iGem team meetup (hosted by Edinburgh) where we introduced our projects and learnt from the challenges each team had faced. This meetup was so helpful in fact, that we hosted a Biomanufacturing discussion where we had attendance from teams such as Vienna (shown in the image below). Science is built off a foundation of sharing information and bouncing ideas off each other; so talking to a team in a similar situation affirmed us of our project and its impacts.

Environment and Social Impact

With our project discussing the alternative to using citrus material to extract our products, considering the environmental impact of our project at a global and local level was extremely important. Despite not getting through to any companies specialising in limonene or PA production, we had a short conversation with Ginkgo Bioworks, a very large American biotech company, who told us about some of the benefits of using a synthetic biology approach in a large-scale industry. At a global level, our project would (in practice) reduce the land, resources and time required to produce PA and limonene in comparison to conventional extraction methods. It is also a much better alternative as it will have higher yield than the conventional extraction that has extremely low peel to essential oil yield. Not having to rely on the growth of plants and use of pesticides /chemicals allows year round growth. This, alongside removing dependence on the citrus industry and being able instead using more common raw materials (like glycerol/glucose) to produce limonene and PA, is why our product would be very beneficial (Jongedijk et al., 2016). As it stands, current research into intracellular limonene/PA production is too inefficient to be used in industry and with limited testing or concrete results, we don’t think our product will be used. However, we hope that our product can serve to show one or two methods that may improve production and that future companies and iGem groups interested in limonene or PA can consider our ideas.

Thinking on a more local level, we were concerned that our product may displace farmers who relied on using their citrus fruits (or lavender) to supply the production of limonene and PA. Vocalising these concerns with Dr Graham Teakle proved immensely helpful, as we confirmed that normal extraction procedures are very inefficient (only yielding small amounts) compared to biomanufacturing. In terms of its impact on farmers, it is much more lucrative to sell their products to larger industries (such as food/juice) and we found there are essentially no farmers focussing on supplying to manufacturing companies. As a matter of fact, improving accessibility to limonene and PA by increasing the production may benefit smaller industries that use it in their variety of products. Both Dr Teackle and Dr Rober Lillywhite mentioned doing a life-cycle analysis (LCA) of our product to better determine its environmental impact compared to the usual extraction methods.

Scientific Impact

When first trying to figure out what project to do and what to research, we were quite overwhelmed with where to even begin. After considering our strengths as a group and everyone's areas of interest, we decided to venture into the production of monoterpenes that have a lot of interest as anticancer drugs. We had the initial idea of wanting to mitigate some of the toxic effects of limonene and PA (produced through the mevalonate pathway) but were unsure where to start with thinking of solutions. With a lot of our team members lacking experience, we wanted to make sure that we designed our project using input from a variety of sources.

One of the first people we got into contact with was Dr Deborah Brotherton, a very experienced consultant who has a vast knowledge of E. coli strains. One of the first things we discussed was using E. coli over S. cerevisiae as a host to our production pathway because it has higher natural resistance. Despite this, because of significantly longer growing times and the short time frame of our project, we settled on E. coli. Within the first few weeks of our summer beginning, we had collected 5 strains of E. coli (some supplied by Dr Brotherton and ours from our supervisor Dr Fabrizio Alberti).

With potential host strains collected, we instantly buckled down to flesh out our project as soon as possible. One of the people who accelerated this process immensely was Professor Tim Bugg (shown below) from the Warwick Chemistry Department. Despite not being his main area of expertise, Professor Bugg gave us great insights as to why specifically perillyl alcohol and limonene may be toxic to bacterial cell membranes. Soon after we had a conversation with Dr Ann Dixon (shown below), learning a little more about membrane stress and potential experiments we could use to detect it. Using this knowledge, we knew that our products must induce large amounts of membrane stress before killing this and had an idea to use this to our advantage…

After doing a little more research, the team came up with two main ideas to reduce toxicity. The first is a membrane pump that will be upregulated in response to membrane stress and the second is a feedback system to shut off production of mevalonate enzymes in response to membrane stress. We next consulted Dr Robert Spooner who gave us a lot of insight and affirmation into our pump idea and proposed the idea of a Lac feedback system to downregulate enzymes. After considering the lac system for a while, we spoke with Professor Orkun Soyer to get his opinion. One thing mentioned in our conversation was the use of Tet to carry out our feedback mechanism instead of lac, and we managed to find the Tet repressible promoter and Tet protein sequence from the iGem parts registry (see more in parts). To round off our preliminary research we spoke to Dr Matthew Jenner who has experience solving biological problems. In his opinion, our pump system was much more promising and simpler to put into place than our Ptet feedback method; because of this, we planned to test each idea out individually as well as together in our upcoming experiments.

With the foundation of our project laid, we next had to plan our experiments. While experiments such as limonene/PA growth assays we had thought of already we wanted to know if there were any routine experiments worth performing. Talking with Dr Munehiro Asally was extremely helpful in this regard. He was the first to tell us about IPTG assays that could test how induction of the plasmid affects growth compared to the production of products (our graphs are seen on the results page). Additionally, further experiments such as using fluorescence to track membrane stress seemed like something we would be interested in. A further conversation with Professor John McCarthy, an expert in molecular system biology, was extremely helpful in informing us how to design our parts and taught us about the many different approaches we could have taken instead.

After a few more weeks of deliberation, we had our parts, experiments and end goal planned out. We decided to use GCMS to detect our compounds (Thanks to our advisor Mabilly Cox Holanda de Barros Dias and to Dr Lijiang Song for testing our samples) and performed many of our experiments in an overnight incubating shaking plate reader (Thanks to advisor Rhys Evans for testing our samples). See all the results we gathered and the final remarks of our experiments on the results page.

References