For the human practices component of our project, we reached out to various experts in the field of agriculture, food regulation and safety, and biotechnology to learn more about the impact our project will have on the world. We spoke with five different experts during the course of the project. The questions and answers for each interview is below.
Interview 1: Dr. Robert Wick, PhD
UMASS Amherst Prof. of Plant Pathology and Nematology
We decided to speak with Dr. Robert Wick, who has extensive research and knowledge regarding plant pathology, to gain insight into the mechanisms that which cause both human and plant bacteria to gather on plants, along with some commonly used preventative techniques.
Question 1: What are some of the common bacteria involved in contamination of field crops? What are some of the common sources of these pathogens?
Dr. Wick said that bacteria such as E. coli and salmonella are harmful to humans but not to plants. Bacteria harmful to plants tend to cause diseases such as black rot, while human pathogenic bacteria such as E. coli have no visible effect. These human pathogenic bacteria end up on plants from various sources including animal manure, fecal matter from wild animals, and contaminated irrigation/wash water.
When we described the use of antifreeze proteins in our project, Dr. Wick suspected that their mechanism of action is a disruption of lipid membrane composition, leading to an antimicrobial effect. He also advised us to be wary of the level of expression of antifreeze proteins. Although plants control protein expression and energy consumption robustly, a constituently expressed protein would prove deleterious for the host plant. Dr. Wick also expressed interest in whether the plants we are working with have similar DNA sequences to the genes that encode antifreeze proteins. If so, this may enable better precision of expression. Additionally, he was curious into the typical inducers of these proteins (e.g., photons, temperature drop, etc).
Question 2: Do you see bacterial contamination of field crops as a prominent issue in agriculture?
Dr. Wick said that plant bacterial contamination issues vary more from crop to crop rather than farm to farm. For bacterial diseases in plants, issues tend to be seedborne, with 1 in 10,000 contaminated seeds being potent enough to infect an entire crop field. All seeds are contaminated from the parent plant.
Question 3: Which types of farms are most usually affected (i.e. small farms, large farms, organic farms, non-organic farms), or does it depend on the farm and situation (i.e. any patterns)?
Dr, Wick described two common modes for plant infection: buying seeds that are already contaminated or growing a crop in a site that had a bacterial disease the previous season. Many contamination issues are governed by weather patterns, with rainy conditions encouraging greater bacteria proliferation than in drier conditions. Water is a breeding ground for bacteria and wind can carry bacteria across a field. When asked which types of farms are most at risk for contamination, Dr. Wick reiterated that plant bacterial diseases are plant-specific and highly dependent on weather. Dr. Wick also mentioned that rotating crops every season helps forestall the spread of contamination, as most bacterial infections arise based on the host infected. For example, black rot of the cabbage family occurs in members of the cabbage family but not in unrelated crops.
Question 4: What is being done to control bacterial contamination of field crops? What are some of those proposed solutions? Is there any progress yet?
Dr. Wick mentioned that while sophisticated tests for human pathogens exist, tests for plant pathogens are less commonly used and not as accurate. When buying a batch of seeds, any bacterial growth proves too minuscule to detect. You cannot assume that a batch of seeds is completely disease-free. In addition to bacteria spreading by a rainstorm, farmers can spread contamination by hand when handling fresh crops.
Regarding conventional ways to prevent contamination, antibiotics are utilized but not as often. Copper can be used to kill bacteria, but it is often ineffective and is washed off by rain rather quickly. Methods of protecting plants are few and farmers exercise caution in practice: they attempt to limit bacterial spread by not picking crops right after a rainfall.
Question 5: What are some the of the biggest challenges facing our proposed solution? Would it be viable economically and obtain regulatory approval? Do you think farmers would mostly express approval or disapproval of our solution, or would they be indifferent? What is your personal opinion on our project?
Dr. Wick said that both he and other farmers would be behind the idea. Some of our project ideas are troubling to him, however. He is concerned that expressing antifreeze proteins all of the time could be too energetically taxing to the plants and disrupt their normal metabolism, but then again it might not hurt them. He recommends looking into how these proteins are induced and how they affect the plant's growth overall.
Interview 2: Dr. Patricia Stapleton, PhD
WPI Director of the Society, Technology, and Policy Program
We spoke with Dr. Patricia Stapleton, food safety and regulation expert, about some of the terminology and regulation involved with marketing a genetically-modified food product.
Question 1: In what case is a “new/genetically modified” crop considered “substantially equivalent” to an existing, unaltered crop? For example, which of the following would be considered transgenic, genetically modified, or natural?:
- In the case of taking a gene from a tick (or any other insect or mammal) and subcloning it into a crop
- In the case of taking a gene from another plant and inserting it into a crop
- In the case of spraying a protein derived from an insect, mammal, or another plant onto a crop
- Does the criteria vary from crop to crop and from situation to situation?
Dr. Stapleton said that regulations vary from country to country, and sometimes from crop to crop. U.S. regulators look at the item to be modified and the potential modifier. If both the modified item and the modifier are well-known, any new crop is considered “substantially equivalent.” For example, carrots and tomatoes are both regulated and well-known, so a transgenic plant comprised of both carrots and tomatoes is allowed.
She also said a transgenic, or genetically-modified (GM) organism is created from any procedure involving the placement of genes from one species into another. Based on this definition breeding is not considered genetic engineering, only laboratory procedures. Any plant deemed transgenic is not considered organic. Surprisingly, there is a list of pesticides and herbicides that can be used on organic plants, but under heavy regulation.
Question 2: What are some the of the biggest challenges facing our proposed solution? Would it be viable economically and obtain regulatory approval? What are some regulations we would have to consider?
Dr. Stapleton said trying to pass a spray would be more difficult because it is considered an application and falls under a different set of regulations. This will entail a battery of tests (e.g. soil tests for environmental impact). We would have to work closely with the EPA and FDA with this type of solution because the environmental and public health risks are higher. In contrast, we would face fewer regulatory hurdles with the transgenic or genetically engineered option. The government has been working with transgenic plants for a long time, and with this experience comes leniency towards solutions in this category. If we do consider employing either solution, Dr. Stapleton recommended that we work with a large agricultural company, as they have the framework, experience, and equipment available to accelerate the approval process.
Question 3: What is the difference between cisgenic and transgenic?
Cisgenic plants have been created for thousands of years through selective breeding. Cisgenic refers to the breeding of two of the same species of plants, and is not referred to as genetic modification by regulatory bodies. Transgenic describes laboratory genetic modification between species of plants.
Question 4: What is the overall view of farmers and consumers on GMOs and transgenic plants? Are they mostly supportive, disapproving, or indifferent? Are there misconceptions? Does it matter to the larger public?
Farmers may be opposed to a spray solution because it could be toxic to their crops. A transgenic solution could be more likely to garner farmer support, especially using the carrot antifreeze protein (as opposed to the tick). Regarding consumers, they often only change their behavior when something has gone catastrophically wrong. In general, consumers don't seek out in-depth information about where their food comes from or what they are eating until a negative event draws their attention to it. People who have had food poisoning might be more likely to try our solution and weigh the options.
Question 5: What is being done to control bacterial contamination of field crops? What are some of those proposed solutions? Is there any progress yet?
There are many challenges facing farmers with regard to foodborne illnesses. Some common sources of contamination include exposure to water runoff, wild animals, fertilizer, manure, applications, and handling by farmers. Once contamination is found however, it is difficult to determine where in the supply chain it occurred (i.e. field, point of harvest, during processing, packaging, transport, supermarket, at home or restaurant). Although it is difficult to prevent contamination, it is easier to prevent exposure, by having fewer people involved and limiting the transportation time of produce. She said that contamination will most often occur in the field.
Question 6: You mentioned to us that about 80% of all GM crops are modified to make them resistant to herbicides, and are likely transgenic from bacteria. What do you mean by this? Do you mean that bacteria are used to make these plants transgenic? Is this common practice?
Dr. Stapleton said she was referring to using genes from bacteria in transgenic plants. Bacterial genes have been used for a long time and are highly-regulated.
Interview 3: Dr. Susan Roberts, PhD, WPI Chemical Eng.
Michelle McKee, Graduate Student, WPI Biology/Biotech.
We spoke with Dr. Susan Roberts and Michelle McKee to gain insight into the field of transgenic plants and possible considerations for our gene gun. Both Dr. Roberts and Michelle have performed extensive research in transgenic plants. Michelle has also extensively used a particle bombarder (aka gene gun) in her laboratory.
Dr. Susan Roberts
Question 1: Which plant cell species do you typically work with when using a gene gun? You typically work with plant tissue cultures, correct? What is the reasoning for working with a plant tissue rather than an entire plant itself?
Dr. Roberts: We work with Taxus plant cell cultures that produce the anticancer agent Taxol. We aim to engineer the cell cultures for various objectives, including increased Taxol production. We use cell cultures because that is the system used commercially to synthesize Taxol.
Question 2: Most of our project work has been with bacteria and using bacterial promoters. What are some of the typical plant promoters that you use in this line of work? What are some of the requirements/conditions for these promoters? Which promoters would you recommend us using?
Dr. Roberts: There are a range of promoters that you can choose for use in plants. I have attached an article that we wrote a while ago regarding development of a particle bombardment method for transient gene expression in Taxus that describes some of the promoters that work in our system. Michelle McKee, a PhD student in my lab, has perfected the gene gun technique and I’m sure she has suggestions for you.
Question 3: Do you find that a certain number of base pairs works better than others? Is there a sweet spot or a desirable number of base pairs for getting plant cells to express a certain gene?
Michelle McKee: I think the smaller the plasmid is, the better, but obviously you need to have all the important sequences in there. I have heard that if the plasmid is too large, efficiency will go down. As for promoters, we just went with the most basic/common sequence, but the stronger the promoter, the more GFP or marker protein there will be to detect. I don’t know about bacteria, but I have found that bombarding from the correct height/distance has played a huge role in efficiency. Lots of people use particle bombardment for stable transformation – it is just easier if you have a faster growing cell line (Taxus is a bit slow).
Question 4: How many generations would you expect a typical gene to carry on for in your experience?
Dr. Roberts: It can really vary. Generally, the gene gun technique results in transient expression, which may last for months. Although sometimes you can get stable expression. For plants, we typically use agrobacterium-mediated transformation to get stable expression.
Interview 4: Glenn Stillman, Stillman's Farm
New Braintree, MA
We were lucky enough to visit Stillman's Farm in Worcester, MA owned by Glenn Stillman. Glenn was able to show us around his farm and answer some of our questions. Our goal was to learn more about the contamination of crops and see if farmers like Glenn would be in support of our project. Glenn Stillman has a background in microbiology and biochemistry.
We met with the owner Glenn Stillman for about an hour and a half. Glenn had bought the New Braintree farm in 1990 and later adopted a slogan for the farm that encapsulates his responsible growing and environmental practices. He adopted the Conscientiously GrownTM slogan because he was dissatisfied with “conventional” and “organic” produce labels. Conscientiously GrownTM serves as an umbrella term for many different growing practices.
Stillman’s farm does not offer genetically modified (GM) crops for marketing reasons. Glenn has nothing against GM crops personally, but does not believe that the benefits of growing GM crops outweigh the negative publicity they may attract. Regarding sprays, Stillman’s farm only uses select herbicides and synthetics on their produce. Not all crops require the use of chemical sprays, however. Glenn told us that he does not list his crops as organic, but mentioned that organic crops have a list of pre-approved chemicals, many of which he does not use on the farm. With his methods, Glenn said he is able to achieve 6-10% organic matter in his soil, an amount higher than typical. The final component of the farm’s Conscientiously Grown practices includes an awareness of local wildlife and the environment by refraining from using toxic organic and traditional sprays.
Sources of Contamination and Washing
Glenn listed two potential sources of bacterial contamination on his farm: manure and wash water. Glenn stopped using manure after he read a story about contaminated spinach. Apparently, some consumers got sick from bacteria taken up by spinach roots from surrounding manure. This type of situation is particularly bad because bacteria that is present inside the spinach cannot be washed off or easily detected. As a result of this, Glenn opts to not use manure on the farm.
For washing produce, Glenn uses water that is treated with bleach. By law, Stillman’s farm is required to have their wash water tested once a year, which has never tested positive for bacteria. Regarding the washing process, produce is picked daily, rinsed and dipped once in water, and is sold. Glenn said that washing not only on the farm but at home, is extremely important for preventing contamination. Bacterial formation is highly dependent on location and transport. For example, crops sold by Stillman’s farm go directly to the consumer, so there is less time for the bacteria to grow. Meanwhile, produce that is shipped by large distributors across many states provides bacteria ample time to proliferate. While it is unlikely that a small amount of bacteria will make an otherwise healthy individual ill, individuals with compromised immune systems, young children, and the elderly are at a higher risk of contracting an infection. For shelf life, Glenn said that produce can sit for up to ten days before it is sold!
Farming Practices for Lettuce
Glenn first germinates lettuce seed in a greenhouse under controlled precipitation/moisture and heat. The seed is germinated under controlled conditions to help it survive and manipulate its growth. Usually, they are germinated in synthetic soil with a high organic composition because traditional field soil would harden up. After germination, the lettuce is transplanted and transferred outside where it hardens up in plastic-lined raised rows before it is picked and sold.
Opinion on Our Proposed Solution
Glenn said he wholeheartedly supports our proposed solution. He said that many farmers would likely be in support as well. Farmers would appreciate the peace of mind our solution would provide, knowing that their crops would not be contaminated and make anybody sick. Knowing the success of the farm is totally dependent on the success of the harvest is a constant source of stress for many farmers. If a harvest is poor or somebody gets sick, it could spell disaster for a farm for years to come. Our solution not only would help prevent illness, but protect farmers’ livelihood.
Some Pictures from Our Trip
INTEGRATED HUMAN PRACTICES
We were fortunate to speak with so many experts in the fields of food safety and regulation, biotechnology, and agriculture. One of the main goals of our project was to use the information we learned from our interviews to better understand the impact of our work and its effect on the world. This section details some of the ethical, social, and environmental implications of our project and how our project changed because of them.
GMOs are an extremely polarizing topic right now. There are some consumers who are indifferent to where their food comes from, while others are more cautious. Many consumers refuse to buy GM food products for both health and ethical reasons, instead opting to buy all-natural or organic ones. One of the most challenging aspects of our project was trying to sort through all of the terminology and regulation involved in GMOs, so we sought the assistance of industry experts. From our conversations with Prof. Stapleton and Glenn Stillman especially, we learned that much regulation of GMOs, including the terms "natural" and "organic", are both ambiguous and misleading. For example, although the term "organic" sound like no sprays or pesticides are used, there is actually a pre-approved list of chemicals by the FDA. According to Glenn, some of these organic sprays are just as scary as the non-organic ones. Glenn doesn't like to use some of the organic sprays because he is worried about their effects, even though he could legally use them.
When it comes to marketing GM food products, we learned that consumer psychology plays a big role. For example, if we were trying to sell lettuce that is genetically-modified to be protected against bacteria, someone who has suffered from food poisoning might be more likely to buy our product than somebody who has never had food poisoning before. There is also an ethical and health component to consumer psychology as well. There are many consumers who believe that GM foods pose certain health problems. In addition, some consumers believe that genetic engineering is irresponsible behavior.
We also found it interesting why Glenn chooses to not grow GM crops on his farm. While Glenn is not personally opposed to having them on his farm, he decided against them for marketing and public relations reasons. Glenn does not believe that the benefits that GM crops could provide to his farm outweigh the potential public outcry and scrutiny he could face. It was a more economically sound decision for his business to sell non-GM crops. If we were going to develop and market any type of GM product, these are all things we would have to consider.
How Our Project Changed
One of the initial goals of our project was to develop a spray to protect crops against bacteria. We figured a spray would require less work than making a transgenic plant and might be more worth our time developing. We hoped to create two different types of sprays: the first being comprised of either antifreeze proteins or curcumen, and the second being comprised of harmless bacteria or bacteriophages that expressed antifreeze proteins or circumen. Both types of sprays would be used as a preventative for bacterial biofilm formation on crops. We were initially very excited with this idea, but didn't know any of the regulatory or social aspects it would entail.
Speaking with Dr. Patricia Stapleton and Glenn Stillman played a large role in determining the direction of our project. Dr. Stapleton told us that a spray would be difficult to get approved and would involve many hurdles. Because a spray is considered an application in agriculture, it falls under further regulation and testing than traditional genetic modification of plants. Genetic modification of plants has been used and studied extensively by the US government, so it would require less work to be approved. Conversely, if we chose to develop a spray, we would have to work closely with not only the FDA, but the EPA as well because we would have to monitor its environmental effects (ie. soil testing). Dr. Stapleton said that a spray will likely have more health and environmental effects when compared to a transgenic plant. As a result of this, farmers are more likely to support a transgenic plant. When we spoke with Glenn Stillman, he shared a similar sentiment. Glenn said he personally tries to refrain from using sprays as much as possible, and other farmers feel the same way. When asked which types of antifreeze proteins we should use (ie. an antifreeze protein derived from a carrot or a tick), both Dr. Stapleton and Glenn said that the carrot antifreeze protein would have a lesser health and environmental impact. They explained to us that farmers are more likely to support proteins derived from plants, as opposed to those derived from animals or insects. In addition, it is easier to get a protein approved that is derived from an existing and commonly eaten crop/organism. Using proteins from organisms that we know a lot about and already consume is less of a health and environmental hazard.
As a result of these interviews, we decided to shift the focus of our project. Rather than looking into only developing a spray, we decided to go the route of genetic modification of plants. Inspired by the recent outbreak in romaine lettuce, we chose lettuce as our plant of choice. Our goal was to show that we could transform lettuce to express both antifreeze proteins and curcumin and show decreased biofilm formation. In lab, we first tried to develop a method of growing and quantifying biofilms on lettuce leaves. Once we were comfortable growing biofilms, we had to transform lettuce leaves with plasmids containing antifreeze proteins and curcumin. To do this, we decided to create our own low-cost gene gun, adapted from the 2016 Cambridge iGEM team's design. Not only was our gene gun able to effectively fire gold particles into lettuce, but it was able to transform lettuce leaves with GFP! In the future, we would hope to transform lettuce with plasmids containing genes for antifreeze proteins and curcumin. If were were able to do this, this could potentially be applied to plant seeds. Based on the advice from Dr. Stapleton and Glenn Stillman, we would only focus on antifreeze proteins derived from commonly eaten and regulated plants to limit their health and environmental impact.