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