Team:Purdue/Design
Aim:
- Select a PAMP that would induce an immune response that effectively combats M. oryzae
- Identify suitable chassis
- Engineer expression of PAMP in selected chassis
- Confirmation and characterization of PAMP expression
PAMP Selection:
We found that chitin was a fitting PAMP for our purposes. It is well documented in the literature that chitin induces an antifungal response in Rice and other plants.
We found that chitin was a fitting PAMP for our purposes. It is well documented in the literature that chitin induces an antifungal response in Rice and other plants.
Chassis:
The following criteria were considered when selecting a chassis.
P. fluorescens st. F113 was an excellent strain for our purposes. Unlike many other Pseudomonads, P. fluorescens is non-pathogenic and readily inhabits most surfaces on rice plants. By inhabiting all surfaces of the rice plant, we are able to insure that PTI can be induced at any point on the plant. Additionally, we were able to find literature that contained a backbone and several promoters that have been used in P. fluorescens.
The following criteria were considered when selecting a chassis.
- Non-pathogenic
- Able to inhabit the Phyllosphere
- Existing tools for expression
P. fluorescens st. F113 was an excellent strain for our purposes. Unlike many other Pseudomonads, P. fluorescens is non-pathogenic and readily inhabits most surfaces on rice plants. By inhabiting all surfaces of the rice plant, we are able to insure that PTI can be induced at any point on the plant. Additionally, we were able to find literature that contained a backbone and several promoters that have been used in P. fluorescens.
Engineering Parts:
Upon investigating the metabolic pathways of the chassis being used it was discovered that P. fluorescens and E. coli had all the prerequisite genes for chitin production with the exception of a gene from chitin synthase. Purdue iGEM chose to use NodC from Rhizobium leguminosarum which is a chitin synthase homolog when designing parts for chitin production.
As proof of concept, we decided to produce chitin in E. coli as it is a well-known organism that is commonly used in synthetic biology. For expression in E. coli, we used a pGEM backbone with a J23100 Anderson promoter, NodC, and a DB1 terminator.
Upon investigating the metabolic pathways of the chassis being used it was discovered that P. fluorescens and E. coli had all the prerequisite genes for chitin production with the exception of a gene from chitin synthase. Purdue iGEM chose to use NodC from Rhizobium leguminosarum which is a chitin synthase homolog when designing parts for chitin production.
As proof of concept, we decided to produce chitin in E. coli as it is a well-known organism that is commonly used in synthetic biology. For expression in E. coli, we used a pGEM backbone with a J23100 Anderson promoter, NodC, and a DB1 terminator.
For Chitin production in P. fluorescens, two plasmids were made using the pSW002 plasmid from Wilton et al. paper. These plasmids contained either a PpsbA or a Pc promoter followed by a NodC optimized for P. fluorescens and a T4 terminator. Due to issues with Pseudomonas transformations a version of this plasmid was also made with the pGEM backbone for expression and characterization in E.coli..
Confirmation and Characterization:
In order to characterize chitin production we used a calcofluor white stain on colonies transformed with the plasmids described above. Calcofluor white is a stain that fluoresces blue when in the presence of chitin and exposed to UV light.
In order to characterize chitin production we used a calcofluor white stain on colonies transformed with the plasmids described above. Calcofluor white is a stain that fluoresces blue when in the presence of chitin and exposed to UV light.
References
- Wilton, R., Ahrendt, A. J., Shinde, S., Sholto-Douglas, D. J., Johnson, J. L., Brennan, M. B., & Kemner, K. M. (2018). A New Suite of Plasmid Vectors for Fluorescence-Based Imaging of Root Colonizing Pseudomonads. Frontiers in plant science, 8, 2242. doi:10.3389/fpls.2017.02242
