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Abstract

Aflatoxins are highly toxic secondary metabolites produced by a number of fungal species in the Aspergillus genus. Of the twenty known aflatoxins, aflatoxin B1 (AFB1) is considered to be the most toxic as it was recently categorized as a Group I carcinogen by the International Agency for Research on Cancer. In Georgia, AFB1 contamination in peanut plants is a major public health issue that is responsible for health complications such as hepatotoxicity and immunotoxicity. As such, an inducible expression system that can efficiently isolate aflatoxin affected plant was engineered using the Gal4/UAS system and single-chain variable fragments (ScFvs). The Gal4/UAS system consists of the Gal4 transcriptional factor (from Saccharomyces cerevisiae) and an upstream activating sequence (UAS) that enhances a minimal promoter for a gene of interest. The binding and activating domains of the Gal4 transcriptional factor were each fused to a unique ScFv that is specific to AFB1. Upon exposure to AFB1, each ScFv will bind to AFB1 and allow for the Gal4 transcriptional factor to be fully activated, which in turn will lead to activation of the UAS and subsequent expression of a downstream gene. The primary gene of interest was BS3, an apoptotic initiator that causes cell death in plants. BS3, as opposed to GFP, confers a greater advantage to this system because the response is easily measured and does not require any additional equipment. All in all, the Gal4/UAS system combined with ScFvs is a novel idea in the field of plant pathology and holds great potential to enhance peanut production within Georgia.

Background

Peanut Industry in Georgia

The main focus of this year’s project is on the regional peanut industry. The subtropical climate and sandy soils in Georgia provide ideal conditions for the large-scale production of peanuts. [1] According to data collected by the USDA, peanut production in the United States was estimated at a record high of 7.23 billion pounds in 2017 (up 30% from 2016). Out of all the peanut producing states, Georgia alone accounted for 50% (~3.61 billions pounds) of the peanut production (Figures I-II). [2] Additionally, the Georgia Peanut Commission reported that the peanut industry contributes around $2.2 billion to Georgia’s $71.3 billion agricultural industry and provides over 50,000 jobs to people across the state. [3-4]

Figure I

Aflatoxin

From avoiding pests to battling infection from deadly mycotoxins, the peanut industry faces numerous challenges not only in Georgia, but also across the globe. One such issue is the contamination of peanuts by aflatoxin B1. Aflatoxins are highly toxic secondary metabolites produced by the fungal species Aspergillus flavus, A. parasiticus, and A. nominus. [5] There are more than 20 known aflatoxins, but the four main ones are aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), and aflatoxin G2 (AFG2). [6] Among these four, AFB1 has been identified as the most toxic and was recently categorized as a Group I carcinogen by the International Agency for Research on Cancer (IARC). [7-8]

In humans, aflatoxins are known to specifically target the liver, but continued exposure to them can lead to a variety of health complications such as hepatotoxicity, teratogenicity, and immunotoxicity. [9-10] Studies have also shown that approximately 18-22% of Georgians are at risk of being victimized by hepatotoxicity. [11] Early symptoms of hepatotoxicity by aflatoxin include fever, malaise, anorexia, abdominal pain, vomiting and even hepatitis. [11] Additionally, aflatoxins have also been found to decrease the overall immunity in children, thereby increasing their chances of infection. [12] As such, because of its severe health repercussions, aflatoxin was on the Rapid Alert System for Food and Feed (RASFF) of the European Union in 2008. [13] Nevertheless, although liver damage by aflatoxin is an important issue, limited doses of aflatoxin are not harmful to humans because various factors - such as age, sex, species, and diet - control aflatoxin toxicity. [14-15].

In addition to its public health consequences, aflatoxin contamination is responsible for severe economic repercussions across the globe. In the USA, contamination by aflatoxin cost producers an average income loss of over $100 million per year [16]. Likewise, in Africa, aflatoxin contamination resulted in a net loss of over $750 million annually [16].

The fungi that produce aflatoxin thrive in warm, humid regions and are commonly found on maize, peanuts, wheat, cotton and tree nuts. [17-18] Recent studies have linked aflatoxin contamination to unhygienic environmental conditions and poor procession; contamination is most frequent and serious at the storage and processing levels along the marketing chain. [19] Consequently, aflatoxin is present in various processed foods, including cereals, spices, dairy products, oilseeds, and packaged nuts [20]. People are frequently exposed to aflatoxin when they unknowingly eat contaminated foods (such as peanuts) or consume meat and dairy products that come from animals fed with infected crops (Figure 2).

That said, in order to prevent the numerous health and economic consequences related to aflatoxin contamination, it is crucial that aflatoxin be detected in foods before they are consumed. Thin layer chromatography (TLC) is one of the oldest methods used to detect aflatoxin [21] but more recently, high performance liquid chromatography (HPLC), liquid chromatography mass spectroscopy (LCMS), and ELISA tests are being used most frequently [22]. However, such detection techniques are highly time consuming and often quite expensive. As such, this year’s project is centered on constructing the basis of a genetically modified peanut plant that averts both the health and economic repercussions of aflatoxin contamination.

Gal4 / UAS & Single Chain Variable Fragments

As mentioned earlier, our primary goal is to invoke some type of response in aflatoxin affected crops . To achieve this end, we designed an inducible expression system that combines the GAL4/UAS system with single-chain variable fragments (scFv’s).

The GAL4/UAS system consists of the GAL4 transcription factor and an upstream activator sequence (UAS) that normally precedes some gene of interest, usually a reporter like GFP. Figure I illustrates this system.

Figure III

Single-chain variable fragments (scFv’s) are merely variants of the classical antibody. Developed by the immune system, antibodies are modular proteins that recognize and bind to specific molecules (called antigens) in an effort to rid the body of foreign pathogens. All antibodies are comprised of four polypeptide chains arranged in the shape of a “ Y “ with 2 “heavy” chains and 2 “light” chains. The base of each antibody is a highly conserved region - in other words, the base of each antibody contains an identical primary sequence of amino acids. However, each antibody is very specific towards a particular molecule due to variability in the upper tips of the “Y” region. scFv’s are recombinant antibodies in that these variable regions of the light and heavy chains are fused together via a linker sequence. This, in essence, optimizes them by increasing their overall stability and affinity for target antigens. [23]

Figure II

All in all, inducible expression systems are ubiquitous in synthetic biology, especially within the realm of the iGEM Competition. This year’s project strives to further strengthen a known expression system, which, in turn, will not only expand iGEM’s existing registry of plant promoters, but also allow future iGEM Team’s to easily express transgene using a binary vector.

Design

This year’s iGEM project was designed as an inducible expression system that combines the mechanism of the GAL4 / UAS system with the versatility of single-chain variable fragments (scFv’s).

In this system, the GAL4 binding domain (BD) and the VP16 activation domain (derived from Herpes simplex virus) were each coordinately fused to a single-chain variable fragment (scFv) that is specific to aflatoxin B1 (Figure III). VP16 was used in place of the GAL4 activating domain because numerous studies have found it to be a stronger transcriptional activator [24-25].

Figure IV

The sequences for both scFv’s were derived from literature on aflatoxin specific antibodies [26-28] and then assembled by our team. Once fused to the corresponding transcriptional unit (either GAL4BD or VP16), we began the assembly of our reporter gene.

Our primary reporter gene of interest was BS3, an apoptotic initiator derived from the bell pepper C. annuum. The BS3 gene encodes a flavin monooxygenase that is activated by AvrBS3 and subsequently induces a plant avirulence response. BS3 was engineered with an upstream activator sequence (UAS) directly preceding it.

Figure V

Theoretically, this system should indirectly induce the expression of BS3 in the presence of AFB1. When AFB1 is present, each scFv should recognize and bind to it, activating the GAL4 transcriptional unit.

Figure VI

The now activated GAL4 binding domain will attach to the upstream activating sequence and the activated VP16 will induce strong downstream expression of BS3.

Figure VII

In summary, there are 4 main components of this system:

  1. VP16 - scFv1 (Antibody #1)
  2. Gal4BD - scFv2 (Antibody #2)
  3. 1xUAS - BS3 (Reporter)
  4. Gal4BD - VP16 (control part)

Experiments

Workflow

Before introducing this system into actual peanut plants, we first wanted to test it on a model plant organism. Nicotiana benthamiana, a common tobacco variety, was chosen as the model organism because it is one of the most widely used experimental hosts in plant virology today [29] and is thus very well characterized. As such, we intended to infiltrate N. benthamiana with our system using Agrobacterium tumefaciens, a bacterium that has the capacity to transfer foreign genes in host plant cells. Agrobacterium-mediated gene transformation is possible through the Binary Vector System, in which a binary vector (a vector that can be utilized by both plants and bacteria) works in conjunction with a helper plasmid (usually the “tumor-inducing” plasmid, or Ti-plasmid) to transfer genes into a target plant host. Today, most binary vectors contain Gateway cloning sites to allow easy exchange of genes from different cassettes [30]. Consequently, when designing our system, we flanked each part with Gateway cloning sites in addition to the standard iGEM restriction sites (EcoRI, XbaI, SpeI, PstI).

Figure VIII

In an effort to minimize “leaky” expression of BS3 and maximize the expression of the antibody parts, two different binary vectors were used: pGWB1 and pGWB2. Both the antibody parts and the control part were transferred into pGWB2, a binary vector that contains the constitutive promoter p35S, allowing for stronger baseline expression of the antibodies and control. Conversely, the 1xUAS-BS3 part was transferred into pGWB1, a binary vector that does not contain p35S, and thus minimizes excessive background expression of BS3.

Protocols

From performing basic cloning techniques to infiltrating N. benthamiana, we used a wide range of techniques in the lab. Find our SOPs below:

Results

Through our experiments, we were able to further characterize the 1xUAS-BS3 part designed by our 2018 iGEM Team and also introduce a new part to the registry: Gal4BD-VP16. We demonstrated that our control part, Gal4BD-VP16, functions as a highly potent transcriptional factor that is able to drive the expression of a downstream gene of interest.

When doing our infiltrations, we had 5 different spots on the leaf. Here is the Key:

Infiltration Spots - Key

  1. BS3 in pGWB2
  2. Yellow Fluorescent Protein (YFP)
  3. Gal4BD-VP16
  4. BS3 in pGWB1
  5. BS3 in pGWB1 + Gal4BD-VP16

Based on these results, it is clear that the control part works as expected. Ideally, the only spot where any sort of cell death should be present is spot 5. However, realistically, some cell death should be apparent in the spots that contain only BS3, either in pGWB1 (spot 4) or pGWB2 (spot 1). This is due to any “leaky” expression that might be occurring in the plant. In all three trials, the greatest amount of cell death was observed in spot 5, followed by spot 1, and then lastly spot 4. This is consistent with what we expected. We expected the greatest amount of cell death in spot 5 because the Gal4BD-VP16 transcriptional factor should be driving strong downstream expression of BS3. Background BS3 expression should be higher in pGWB2 (spot 1) than in pGWB1 (spot 4) due to the presence of the constitutive p35S promoter. This is evident through spot 1 showing higher levels of cell death than spot 4. Finally, we know that the control part functions properly because there is no cell death in spot 3 (where Gal4BD-VP16 is infiltrated on its own), but there is cell death in spot 5 (where Gal4BD-VP16 is infiltrated in conjunction with 1xUAS-BS3).

Future Directions

The next step of this project is to test our system with the antibody parts in the presence and absence of aflatoxin B1. Currently, we are in the process of getting access to aflatoxin from other researchers on campus and developing an appropriate experimental setup with the correct controls. After testing this system in N. benthamiana, we intend to test it on different varieties of peanut crops and ultimately produce a modified peanut plant that contains in inherent resistance of AFB1. Additionally, given how ubiquitous inducible expression systems are in plant biology, it is our hope that future plant biologists will be able to use the vector systems, reporters, and recognition devices that we create.

Safety

Right-to-Know Training:

The Right-to-Know Training informed about the basics of the Right to Know Law. The training provided an understanding of the Hazard Communication Standard label elements and Safety Data Sheets.

Hazardous Waste Training:

Hazardous Waste Training was required for all who accumulated or generated hazardous waste. The training informed about the safe handling and use of hazardous wastes, including training on certain chemicals, equipment, and personal protective equipment.

Bloodborne Pathogens Training:

Bloodborne Pathogens Training was required for anyone who would be later exposed to blood or potentially infectious material. The training informed on basic information about bloodborne pathogens, including common modes of transmissions, identifying hazardous labels, and preventing exposure.

UGA’s Lab Safety Basics/Safe Secure Science:

Lab Safety Basics Training/Safe Secure Science was required by all UGA student researchers. The training provided an overview of basic laboratory practices and maintaining a safe working environment.

BSL-2 Online Training:

BSL-2 Online Training provided information on the required safety protocol utilized in a Biosafety Level 2 laboratory. The training also provided a basic understanding of the health risks associated with the infectious material used in the laboratory.

Proficiency for Standard and Special Microbiological Practices:

The Principle Investigator, Brian Kvitko, required all personnel to be proficient in the daily work procedures used in the lab. All personnel was sufficiently trained to maintain a safe working environment.

Kvitko Lab On-Site Safety Training:

The lab manager, Amy Smith, provided on-site safety training for both the general and specific protocol used in the Kvitko Lab. The training also identified the locations of all equipment, waste containers, and safety materials.

Kvitko Lab Practical:

The Principle Investigator, Brian Kvitko, required all personnel to be tested on maintaining sterile techniques in the lab. Each researcher was tested on bacterial inoculation, 3-phase plating, and lawn plating.

Miscellaneous Safety Information:

All wastes and equipment were sterilized and autoclaved after use. Ethidium Bromide could also be found in the lab, and contact was avoided with the mutagenic agent. Although aflatoxin B1 has not been utilized yet in our experimentation, new protocols will be developed and safety training will be administered for the proper and safe use of aflatoxin B1.

References

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