BMBMethod/Oct21Minutes

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Karla

  • Engineer other bacteria to photosynthesize (bacillus and e coli)
  • Find transformation system for Heliobacteria
    • Anerobic jar’s don’t work great, little room for plates
      • Stack w/stick or paper towel roll to have more plates per jar
    • Heliobacter grow to slow/don’t grow, problems with temp
      • Try top agar or pour agar


Mike

  • Bacolovirus doesn’t want to express NELF, kills them?
    • Try using a signal peptide to target the proteins for secretion to get them away from the cells and hopefully decrease toxicity


Cell-free expression

  • New cell-free expression system with dual rxn setup (separated by membrane). Synthesis rxn and feeder rxn for substrate to diffuse from and inhibitory products to fuse into.
    • Email Doug


Wiki

  • Bede had previously had a good idea about collecting protocols from various labs into a single wiki for us to all have available
    • Golbeck lab now uses qwartzy for their protocol organization
    • Dave controls Gilmour lab’s protocols, but everyone mostly uses their own procedures
    • Joseph is going to set up a wiki within OpenWetWare


Tips/Tricks

  • SLiCE cloning
    • Like InFusion, but is recombination based and (according to the paper) allows you to “loop out” regions during cloning
    • Can make your own enzyme with a very simple protocol from a bacterial strain
      • Joseph obtained strain from original authors
    • Email paper to Brooks, Doug, Karla, Mike
  • Western blotting
    • Can use TBST (0.5% tween) for blocking during western blotting rather than milk/BSA
      • Works for both nitrocellulose and PVDF
  • Competent cells
    • Simple protocol (Inoue et al 1990) for making competent E. coli a ~109 cfu/ug efficiency
    • Email paper to Brooks, Doug, Karla, Mike
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