Project Description

Abstract

This year UGA iGEM has begun a new project to genetically engineer a solution for a regional problem. Our team is working to create a biosensor for aflatoxin B1 (fungal toxin) in peanuts, as our home state produces 45% of the U.S. peanut crop and is the number one producer for peanuts in the United States. Aflatoxin is a major concern for peanut producers as it has serious health implications as a highly toxic and carcinogenic compound. One of the primary ways in which Aflatoxin B1 is controlled or detected in peanut crops is through the physical separation of contaminated peanuts. In the interest of making a cost-effective impact, our team will be creating a biosensor that will brightly fluoresce or change color when Aflatoxin B1 is detected. This will make color sorting and separation of the contaminated peanuts much easier and reduce the economic impact of this fungal toxin on one of Georgia’s biggest agricultural industries. This approach is unique as we will be working to genetically modify bacteria to detect this toxin. This modified organism will be a much cheaper alternative to the status quo, which requires high cost, specialized equipment to sort out the contaminated peanuts.

Background

Riboswitch Background:

Riboswitches are RNA molecules that contain a regulatory sequence that controls translation of the encoded protein. In general, translation is controlled through the sequestration of the ribosome binding site (RBS) from the ribosome, usually accomplished through the formation of secondary structures involving complementary base binding within the mRNA molecule itself. Alternatively, in a two-component aptameric system, this can be accomplished through the use of RNA aptamers and their complementary bases.

A Two Component Aptameric Riboswitch

An aptameric sequence is a nucleic acid sequence with high affinity for a given regulator molecule. Thus, in a two-component system, the regulatory molecule may compete with the aptamer’s cDNA for binding to the aptamer. If the affinity for the regulator is higher than the aptamer’s affinity for its cDNA, the cDNA will be released, thereby allowing translation to occur on the protein encoding strand.

Generation of a Biosensor for Aflatoxin B1

This year, UGA’s iGEM utilized this form of aptameric riboswitch to attempt to create a biosensor for detection of Aflatoxin B1 (fungal toxin) in peanuts. Building off of the work of Wang et. al (2016), we created several RNA-based “expressor” constructs consisting of a constitutive promoter, cDNA to the aptamer, an RBS, and a GFP reporter. We also created a “repressor” sequence by incorporating an RNA version of Wang et. al’s aptameric sequence from his 2016 study along with an anti-RBS and constitutive promoter. We hypothesized that when cotransformed these RNA molecules would be bound using complementary pairing. However, upon the introduction of aflatoxin, the repressor’s aptameric sequence would bind preferentially to the toxin, releasing the GFP transcript to be translated.

The Impact

Our team is working to create a biosensor for aflatoxin B1 (fungal toxin) in peanuts, as our home state is the number one producer for peanuts in the United States. Aflatoxin is a major concern for peanut producers as it has serious health implications as a highly toxic and carcinogenic compound. One of the primary ways in which Aflatoxin B1 is controlled or detected in peanut crops is through the physical separation of contaminated peanuts. In the interest of making a cost-effective impact, our team will be creating a biosensor that will brightly fluoresce or change color when Aflatoxin B1 is detected. This will make color sorting and separation of the contaminated peanuts much easier and reduce the economic impact of this fungal toxin on one of Georgia’s biggest agricultural industries. In doing so, we hope that manufacturing costs of peanuts may be reduced, allowing food supplies at the local, national, and international level to be increased through the production of novel food processing technologies.