Team:CSU Fort Collins/Design

COLORADO STATE
UNIVERSITY iGEM

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Design

Contents

1 Proposed Process
2 Evaluating Our Design
3 Functional Prototype Creation

Proposed Process

The process we have designed is for the use of frying oil waste as a feedstock for E. coli which can use that energy to produce trans-zeatin. We would feed the frying oil waste as well as lipase and other cellular growth components into a vessel where extracellular breakdown could occur. From there, the partially-broken down frying oil would be piped into a continuously-stirred bioreactor containing our E. coli with the enhanced fatty acid breakdown and trans-zeatin production capabilities. Once biomass is sufficient, the cells would be lysed using sonication. Then, large cellular membrane and other components would be removed from the supernatant. The supernatant will then be transferred to a column for solid phase extraction to create the final product. A process flow diagram showing our required equipment and flow streams is below.

Process Flow Diagram showing our proposed process for trans-zeatin production using frying oil waste

Evaluating Our Design

We spoke to employees at the City of Fort Collins to discuss how our process would integrate into frying oil recycling in our community. In Fort Collins, most restaurants pay a company to retrieve and recycle their oil into biofuels. Our process could allow for restaurants to break even or be paid for recycling their spent frying oil. And while Colorado is very eco-friendly, many places in the United States and around the world could benefit more incentive them to upcycle their oil. Our use of frying oil waste as an alternative substrate for chemical production would not necessarily replace, but expand the amount of frying oil waste recycled instead of sent to landfills.

The production of cytokinins in bacterial hosts also offers an important alternative to the time- and cost-intensive process of extraction from plant tissues. Our proposed use of biological hosts to manufacture reagents is part of a larger movement in the synthetic biology community to work towards greener production. Ideally, products would be transported outside of the cells. This way, the current standard of using harsh chemicals to extract the product is unnecessary. The cheap production of zeatin would also allow for its expanded use in industry and agriculture. Because zeatin can be used to increase plant biomass, it has potential applications in food, feed, and fuel.

Functional Prototype Creation

A picture of our bioreactors ready to be set up and inoculated

Our team attempted to build a functional prototype of both the trans-zeatin production and frying oil breakdown steps of our process.

We grew our strains with the trans-zeatin biosynthesis pathway in 1 L bioreactors over 72 hours. Bioreactors are often the first prototype created before scale-up can begin on manufacturing processes. While the results of this experiment were inconclusive, we were able to design an experiment which represents a step towards the scaling up of our process.

We were able to show a functional prototype of our frying oil digesting strain. We developed an experiment which grew our strain on frying oil waste (both at 100% and 50% concentrations) donated to us by a local restaurant,The Mayor of Old Town. The results of this experiment showed that our lac promoter:fadD:fadL construct improved the cells’ ability to grow on actual frying oil waste.

We tested strain growth in 100% frying oil (left) and 50% frying oil (right) solutions.