This summer I had the amazing opportunity to work in the Bartlett lab on a project that connects an agriculturally important grass trait with molecular biology. My goal was to create a triple mutant in the model grass Brachypodium distachyon, with alterations to all three SHORT INTERNODES (SHI) transcription factor genes. I began the summer by selecting spacer sequences that I would use to target the three B. distachyon SHI genes. I chose two targets per gene that were approximately 200 base pairs (bp) apart, hoping for a 200 bp dropout in each gene that would seriously inhibit the gene products in my triple mutant line. I had success assembling the six spacer sequences into “Level 1” plasmids with promoters and guide RNA, but ran into issues with linking them back to back in a “Level 2” plasmid. The plasmid assemblies are done using restriction enzymes that leave sticky ends for reannealing once the temperature is lowered. However, the presence of an extra restriction enzyme site in the backbone of the Level 2 plasmid has halted my progress this summer. I will continue to work at this problem over the fall and hopefully have a mutant by the end of the year!
