System Design
One of our big issues to overcome was how to trigger RNAi production for gene silencing in response to drought conditions. We didn't want bHLH61 knockdown to be occuring all the time, therefore we researched heat-inducible promoters in the iGEM registry and found iGEM Part BBa_J100387. We used this sequence to create primers (BBa_25RWK0XJ and BBa_25ESH2UM) to replace the constitutive promoter on pSB1C3 with the DnaK promoter making our new plasmid heat sensitive and hopefully siRNA production occuring only under high heat conditions.
Wet Lab Results
Wet lab work focused on engineering the pSB1C3-tsPurple backbone to have both the DnaK promoter and siRNA sequences. First, plasmid DNA was successfully isolated by mini prep(196 µg/mL). Using NEB Q5 mutagenesis, siRNA sequences targeting GFP or bHLH61 were introduced. Agarose gel analysis indicated that the expected 100 bp size increase was not observed, suggesting the mutagenesis step requires troubleshooting.
Experimental protocol and lab book can be found here.
Plant Growth
We started by germinating Arabidopsis thaliana lines carrying GA-YFP, ER-YFP, SEC-RFP, and AFVY-RFP reporter genes to establish proof-of-concept models for RNAi knockdown. Of these, the SEC-RFP and AFVY-RFP variants showed the most consistent growth and will be used for future silencing assays.
Next Steps
Our immediate priority is troubleshooting the siRNA mutagenesis step, repeating PCR with optimized primer design and cycling conditions to confirm insertion of promoter and siRNA sequences into the pSB1C3-tsPurple backbone. Once constructs are validated by agarose gel and sequencing, they will be transformed into E. coli for functional testing of the heat-inducible dnaK promoter using the tsPurple reporter. Parallel experiments will use in vitro transcribed siRNA on Arabidopsis reporter lines to establish proof-of-concept gene knockdown assays. Fluorescence imaging and qRT-PCR will quantify silencing efficiency. Following successful validation in Arabidopsis, we will move to testing bHLH61-targeting siRNAs in canola seedlings. In addition, development of an auxotrophic Arthrobacter globiformis chassis strain will enable safe delivery of engineered constructs in seed coatings. Future work will also expand the computational models by incorporating plant growth and drought-stress parameters to predict field-level performance, ultimately guiding the design of a drought-tolerant canola system.