Additional research on Rhagium inquisitor antifreeze protein and gene synthesis options
Wednesday:
Tokyo Tech iGEM BioBrick BBa_K193209 arrives, Davies Lab type III AFP (Zoarces elongatus) arrives, transform and streak plates with respective genes
Completed modelling/homology studies for RiAFP, but failed to determine any sort of homologous secondary protein structure
Meeting with faculty adviser Prof Farren Isaacs about project and RiAFP gene synthesis design.
Emailed various companies for sponsorship for gene synthesis
Emailed various professors for suggestions for AFP extracellular export mechanism
Emailed other iGEM teams for sponsorship suggestions
Thursday:
Retrieved Origami cell lines (for better disulfide bond formation in cytoplasm, allowing better protein folding) from Yong Xiong lab
Began designing various permutations of RiAFP gene, with possible His-tag, GFP fusion, pelB/OmpA export leader sequence, etc on IDT website after securing sponsorship
Friday:
Decided on IDT construct with pelB-eGFP-TEV-RiAFP-His and ordered gene from website
Contacted Fass Lab of the Weizmann Institute about various TmAFP plasmids to compare against Tokyo Tech BioBrick
Saturday:
Miniprepped Tokyo Tech plasmid and Canadian ZeAFP Type III plasmids and nanodropped for concentration
Subcultured 2 colonies for Canadian ZeAFP-transformed BL21s in 100 mL flasks
Designed primers with BioBrick prefix/suffix overhang for PCR amplification of RiAFP synthetic gene
Sunday:
1 in 40 dilution of previous day's overnight subculture
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