Team:Purdue/Experiments
Our summer project began with preliminary research on how chitin was produced in bacterial cells. When researching how bacterial cells produce chitin, we first had to decide what bacterial vectors we would be working with. We decided upon Pseudomonas fluorescens as the vector we were transforming into, and Escheria coli as the first chassis for the gene of interest. We selected Pseudomonas as our vector because this microbe is commonly found in soil and would be easy to integrate into its natural habitat near rice plants. E. coli was selected as the first organism to transform into because it is commonly used in synthetic biology, and its metabolic pathways are well-defined. We were able to determine that E. coli can produce chitin, but that is not a metabolic pathway that is normally utilized in the wildtype.
We also determined how chitin is utilized by Magnaporthe oryzae, and how plant immune systems work to recognize this product. Chitin is produced across both the outer membrane and inner membrane in bacteria, so we determined that we wouldn’t have to lyse the cells when confirming the product with the calcofluor white assay. The calcofluor white assay was used to confirm the presence of chitin- when calcofluor white binds to chitin, it fluoresces the color blue under a UV light.
Another main facet of our research was understanding the impact rice blast fungus has on rice crops worldwide. We aggregated data from many studies on the economic impact of rice blast fungus and were able to discover that rice blast fungus causes around 70 million dollars in damage yearly. Up to 30% of rice crops are damaged by this fungus every year, and this amount lost could feed around 60 million people. Since rice blast fungus has such a large and worldwide economic impact, this only solidified our decision to come up with some sort of counteracting measure.
All of the protocols that were utilized for our experiments can be viewed by clicking the red button below.
