Team:Alberta/Methodology/Notebook/Genetics

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METHODOLOGY

Notebook

Still in the planning stages of our Genetic modifications. Researched the genetic capabilities of Neuorspora, and how we should transform it. Research into possible thioesterases, promoters, terminators, flanking regions, and antifungal selection ongoing. Started our collaboration with Dr. Frank Nargang and Dr. Dave Stewart.
Decided on using tesA’, a thioesterase from E. Coli, as our synthetic gene. Decided to simultaneously knock out FadD by inserting construct in its place. Decided on Hygromycin B as selection method. Decided to use Actin promoter and trpC terminator for high gene expression.
Tried isolating N. Crassa chromosomal DNA via glass bead “crush” method and ethanol/isopropanol precipitation. Results were inconclusive.
Collaborated with last year’s iGEM team, Genomikon, to design our assembly and construction method. Decided on the modified BioBytes 2.0 format.

Went over to Dr. Frank Nargang’s lab, and received detailed instructions in growing and transforming N. Crassa. Learned tips and tricks, and general handling characteristics we should be aware of. Graciously received materials and reagents from them.
Ongoing design of our synthetic gene and all primers. Codon-optimization of tesA’ thioesterase ongoing, as both secondary structure of mRNA construct and %GC content need to be taken into account. N. Crassa codon usage table used to optimize gene, with some exceptions for %GC considerations. A, P, R, and G changed to their second most-used codon. Scanned and removed BsaI cut sites.