Research Projects

Bacopa monnieri is a plant that produces triterpenoid saponins through a complex metabolic pathway. The isoprenoid pathway is responsible for the cyclization of 2,3-oxidosqualene, leading to the formation of triterpene and steroidal backbone. Squalene synthase converts two FPP molecules into C30 molecule squalene, initiating the first oxygenation step in phytosterol and triterpenoid biosynthesis. Squalene synthase catalyzes important regulatory reactions, such as converting squalene to 2,-3-oxidosqualene in the sterol pathway and cycloartenol and lanosterol in the triterpenoid pathway. CRISPR/Cas genome editing can be used to design a Cas protein to target CAS1 and LAS1 to block the sterol pathway. This will increase the productivity of triterpenoid saponins, such as bacoside A and bacoside B, which have high commercial value. The Cycloartenol Synthase (CAS1) and Lanosterol Synthase (LAS1) genes of Bacopa will be amplified and sequenced using specific primers based on common gene sequences from related plants. Analyzed sequence results will be used to design gRNAs and clone gRNAs to target and modify the LAS1 and CAS1 genes of Bacopa. The optimized Agrobacterium transformation protocol will be used for transformation studies. Selected lines will be subjected to PCR and sequenced to confirm modification events at a single nucleotide base level. Bacosides A and B production levels will be compared with control and mutant lines using real-time PCR for quantification.