Researcher(s)
- Samantha Oliver, Chemical Engineering, University of Delaware
Faculty Mentor(s)
- Kevin Solomon, Department of Chemical and Biomolecular Engineering, University of Delaware
Abstract
Establishing Agrobacterium mediated genome engineering of romaine lettuce leaf-surface yeast for human pathogen detection
Current foodborne pathogen detection methods (culturing a sample and screening using PCR) for leafy greens are effective but time consuming. The detection process takes 2-3 weeks, which is a significant fraction of lettuce’s short shelf life. Sampling is also destructive, meaning that only a fraction of each batch can be tested, meaning contamination can go undetected.
Whole cell biosensors are a promising detection option. Whole cell biosensors are cells that have been modified to express a reporter when they interact with a contaminant. These cells can immediately notify if a contaminant is present by changing color and have already been implemented for organic compound and heavy metal detection.
For this approach, we had to consider which species of yeast that naturally occurs on lettuce is the best candidate as a biosensor. Sporobolomyces lactuca is the most abundant species of yeast occurring on lettuce, and flow cytometry indicates it is a haploid species meaning a simplified genomic editing process.
Developing Sporobolomyces lactuca into a biosensor requires a genetic toolkit, beginning with a transformation method to introduce engineered plasmids. The aim of this project is to develop a method to transform Sporobolomyces lactuca using Agrobacterium mediated transformation (ATMT). ATMT utilizes Agrobacterium’s natural system to insert an engineered DNA cassette into a host species. ATMT has already been successful in related red yeasts, including those in the Sporobolomyces genus. To evaluate if ATMT will be successful, we designed novel promoters with varying lengths of the GAPDH gene, which is native to Sporobolomyces lactuca. This is a common upstream testing strategy to identify native promoters. We are designing an experiment using Rhodotorula toruloides as a positive control, and designing plasmids with multiple variations of both Rhodotorula toruloides and Sporobolomyces roseus elements.