Team:Vanderbilt/Project

Introduction

The production of plant essential oils and their derivatives represents an over 9 billion dollar industry when considering just their applications in the food and fragrance industries ¹. A staggering 23 million kilograms of citrus oil alone are produced worldwide each year. Up until only a couple decades ago, the production of these essential oils was done exclusively by chemical extraction from plant material. However, the sudden emergence of synthetic biology a versatile and efficient tool has the potential to transform this immense industry, the products of which nearly everyone will come in contact with on a daily basis.

By harnessing existing biosynthetic pathways and introducing enzymes taken from plants into more maleable model systems, it will be possible to significantly improve on current methods of the active components of essential oils, most notably the terpenoids. While most plants express terpenes in the range of parts per million and thus require very large scale operations to be commercially viable, early forays into the biological production of terpenes have proven that it is possible to improve yields 100-fold ². The first aspect of our project involves using the great potential of synthetic biology to design a commercially-viable strategy for the production of any class of terpenoid.

Just as important to the economic benefit of this approach is its environment benefit. With chemical terpene extraction being such a relatively inefficient process, it is necessary to process large amounts of plant material to get a substantive yield. This may not pose as significant of an issue for citrus growers, but many of the most prized compounds are taken from the rarest species of plant. The continuation of the status quo in terms of terpene extraction is not an environmentally sustainable solution. In our selection of terpenes, we placed a large emphasis on choosing compounds from some of the most rare species possible.

The best example of this idea behind our project can be seen in the gene for santalene synthase. The only species know to have genes to produce this terpene are found in remote regions of India and Australia, and one of them is listed as a vulnerable species by the IUCN. The trees can live for hundreds of years, but are the target of widespread over-exploitation, to the point that, for example, the Indian government has banned the export of sandalwood. Synthetic biology can produce the exact same active ingredients of sandalwood oil in a way that is both more economically and environmentally sound.

In order for our idea to truly be applicable to exotic and endangered species of plant, we had to take an approach that was quite different from the one most iGEM teams have historically taken. What we were looking for was a quick, inexpensive method of cloning genes that was also compatible with species that have not had their entire genomes sequenced. Often, iGEM teams resort to synthesizing their genes through third parties. However, this can be fairly costly especially given the moderately large size of many of these synthase genes, may take several weeks or even a month to finish synthesizing, and cannot be done unless the gene's sequence is known in its entirety. By going back to basics and taking raw plants as our source material, we were able to avoid these issues and demonstrated how our approach was a practically viable one.

Cadenine Main essential oil: Cade oil Applications: Antifungal, bactericidal, and antioxidant Plant Species: Gossypium hirsutum (cotton) Synthase Gene: δ-cadenine synthase (E.C. 4.2.3.13)	Carene Main essential oils: Rosemary and Cedar oil Applications: Insecticide, anti-inflammatory, and central nervous system depressant Plant Species: Picea abies (norway spruce) Synthase Gene: carene synthase (E.C. 4.2.3.107)	Humelene Main essential oils: Hops oil Applications: Culinary spice, and anti-inflamitory Plant Species: Zingiber zerumbet (shampoo ginger) Synthase Gene: α-humulene synthase (E.C. 4.2.3.104)
Myrcene Main essential oils: Thyme and Hops oil Applications: Fragrance, analgesic, and anti-inflamitory Plant Species: Perilla frutescens (Green Shiso) Synthase Gene: myrcene synthase (E.C. 4.2.3.15)	(R)-Linalool Main essential oils: Lavender oil Applications: Fragrance, and insecticide Plant Species: Mentha citrata (lemon mint) Synthase Gene: (R)-linalool synthase (E.C. 4.2.3.26)	(S)-Linalool Main essential oils: Citrus and Coriander oil Applications: Fragrance, and insecticide Plant Species Chosen: Arabidopsis thaliana (thale cress) Synthase Gene: (S)-linalool synthase (E.C. 4.2.3.25)
Sabinene Main essential oils: Junier Coriander oil Applications: Spice, and ntimicrobial Plant Species Chosen: Salvia officinalis (sage) Synthase Gene: (+)-sabinene synthase (E.C. 4.2.3.110)	Santalene Main essential oils: Sandalwood oil Applications: Fragrance, antiviral, and tumor-suppressant Plant Species Chosen: Santalum album (sandalwood tree) Synthase Gene: α-santalene synthase (E.C. 4.2.3.82)	Zingiberene Main essential oils: Ginger oil Applications: Flavoring and pesticide Plant Species Chosen: Ocimum basilicum (basil) Synthase Gene: α-zingiberene synthase (E.C. 4.2.3.65)

Design

Our project had several co-dependent sub-project t

Methods

Our project had several co-dependent sub-project that were all worked on in parallel. These can roughly be divided into two categories: the first involving work on our synthase genes and the second involving the construction of a new, specially designed plasmid vector. We tried two different team structures over the year to see which would give the best results. For the Spring, we had the original idea of dividing members into independent groups, each working on a specific terpene. Each group was headed by a single group manager who would teach 4-5 new members the protocol that was to be preformed and then supervise that the work was carried out correctly. On occasion either the group managers or the organization president or wetware director would also given lessons to teach members about the techniques and theory involved at each step. All group managers were in turn trained by either the president or wetware director, both of whom had come with the experiment, acquired all the necessary primers and reagents, wrote up the protocol, and had preformed it prior to any group-phase work for the purposes of troubleshooting and predicting where issues may come up. The president or wetware director also helped the group manager in being present during all experiments for answering questions, preparing materials, and other forms of assistance.

Each group first planted seeds under the appropriate soil, humidity, and temperature conditions at the Vanderbilt Greenhouse. Once the majority of these grew into saplings with green leaves, After each group had completed a number of experiments, work began on plasmid construction.

Results and Directions