iGEM Yale

Week 3: May 29-June 5

  • Monday:
    • Prepared LB media and cultures
  • Tuesday:
    • 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