If you’re one of the folks who looks forward to pumpkins every fall, the long summer wait can be tough. For farmers, the summer might become a huge issue if climate change leads to more drought in the crucial growing season.

What if farmers could instead choose to ripen them in June with just the flip of a switch? Groundbreaking Colorado State University research could someday help farmers make that a reality.

Researchers working with Professor June Medford, in biology, and Professor Ashok Prasad, in chemical and biological engineering, collaborated to create a “toggle switch,” using genes that can target specific traits in plants and turn them “on” or “off.” Their interdisciplinary research was recently published in the journal ACS Synthetic Biology.

The genetic toggle switch acts similarly to a circuit in electronics, and is made possible by synthetic biology — the marriage of biology and engineering.

“A genetic circuit is just a big piece of DNA that we have inserted into a plant that allows us to regulate whatever trait we want, such as controlling flowering so that a tomato plant ripens all at once,” said Medford. 

Up until now, genetic circuits have only been used in single-celled organisms. Applying this principle to plants is a little more difficult, said Tessema Kassaw, a research scientist in the Medford lab and a first author on the paper. 

“Part of the challenge is the lifecycle of plants, which have a relatively long lifecycle compared to bacterial systems that genetic circuits have previously been used with,” said Kassaw. “Also, the multicellular nature of the plant — their roots, tissues, vegetative and reproductive organs — makes it complex.”

PAIRINGS

The process began in the Medford lab where Kassaw assembled genetic components — the genes that affect the traits and characteristics of plants — and tested them in singular plant cells. Kassaw made hundreds of genetic components, totaling more than a million possibilities of pairings.

“This is where the math came together with Prasad’s lab because they would tell us mathematically which pairings would give us the best functions,” said Medford. “So instead of testing about a million possibilities, we could just try two or three.” 

Kassaw then translated the data from the Prasad lab into a living plant by injecting it into the plant and seeing if the desired traits could be toggled on and off. The Medford and Prasad labs would go back and forth tweaking the genetic components, testing them in plant cells, quantifying the data, and then testing it in the whole plants. 

“Sometimes working with plants is like watching the grass grow,” joked Medford. 

The team went through a series of toggle switches to strike a balance of the right genes and quantities for the desired outcome. For example, in the 2.0 version of the switch, a trait could be turned on, but it couldn’t be turned off again.

“As biologists, we understand biology really well, but we partner with folks like Ashok Prasad and Wenlong Xu who understand math and engineering, and aren’t afraid to find the signal amid the noise,” said Medford. “In doing so, we now have the ability to make switches.”

Finding that signal amid the noise is where synthetic biology comes in. In this case, it means engineering a living organism to do something different, based on measurement, mathematical modeling, genetic manipulation and many iterations. 

MATHEMATICAL MODELING

The Prasad lab used mathematical modeling to identify two things. First, they had to know the inner workings of normal circuits that control a plant’s biology. Then they could make a synthetic circuit  to make the plant do something different.

After each circuit, the lab measured the results and applied math, over and over again, to make sure that the proteins interacted with one another. Collecting quantitative data is important at this step so researchers can observe whether the protein interacts the way they need it to. 

“Trying it without data and analysis is equivalent to taking a piece of silicon, shoving it into an electronic circuit board and saying, ‘here’s my transistor.’ Obviously, no one does it that way,” said Prasad.

The complexity of living organisms limits how much genetic material researchers can add to a plant, and how they can manipulate it. Wenlong Xu, a graduate student in Prasad’s lab during the research and a first author on the paper, used image processing, data analysis, and modeling to work with the Medford lab on iterations within those limits. 

“Scientifically, the biggest hurdle was convincing myself that quantitative synthetic biology could work in a multi-cellular, long-generation organism,” said Xu. “Personally, the challenge was staying patient and optimistic through the long turnaround time of the plant cycles.”

For Prasad, the step-by-step, iterative process of the research opens up the next challenge: being able to eventually manipulate plants with other types of circuits.  

“What we find is that different types of circuits are suited for different purposes, which is, of course, exactly the way it happens in living organisms,” Prasad said. “Can we control the composition of the fruit by controlling some aspect of metabolism that’s going into the fruit?”

Medford and Prasad describe the research as “a proof of concept,” and expanding how the circuit is used can lead to many other possibilities. 

“In the future, you could see how you could do this using methods to directly edit the plant genome,” Medford said.

This project also offers promising outcomes for the agriculture industry in the face of unpredictable and unseasonable climates. Farmers could control the state of their crops by turning “on” the toggle switch to signal to the plants when it’s time for them to ripen for harvest. If drought altered the fall pumpkin harvest, for example, using the switch could help them grow earlier and retain size and nutrition.

Medford and Prasad joked that one day they could manipulate the astonishingly massive pumpkins seen nationwide every fall in the news, weighing tons and almost the size of a small car. 

“Imagine that you could actually change how carbon goes into those pumpkins, and have a pumpkin that is essentially a giant batch of oil,” said Prasad. “I mean, that would be quite something.”