• Updates from Brian
    • The C. hutchinsonii have arrived!
    • Figured out a way to measure FAEE - High Pressure Liquid Chromatography (HPLC) or Reverse Phase Liquid Chromatography (RPLC)
      • How HPLC/RPLC works - small volumes (~100 uL), high pressures; column binds everything but with different affinities
        • use a pump to create a gradient with 2 different solvents (i.e. water and acetonitrile) to separate by polarity
        • Get a set of peaks (vs times), each of which corresponds to a species
        • Must first take a pure sample of the species of interest to find out where the peak occurs
      • Got a machine for free from a pharma company that we can use
    • Last Thursday, Brian tried recreating the modification of C. hutch in geneious, but he couldn't
      • before, people didn't know the full sequences of the parts that they were working with
      • the paper on C. hutch was like this - they didn't know the exact sequences
      • Now, we must rebuild the 
  • Today - come up with a list of things to do before June 8th:
  1. Team 1 - Grow C. hutch
    1. knowledge base: how-to's for 
      1. liquid + solid cultures
      2. transformation protocols
      3. select
      4. measure growth (Colony Forming Unit (CFU) plates, OD, coulter counter, cytometry)
      5. measure rates of cellulose degradation, rates of byproducts (small organic acids, pentoses/hexoses) production
      6. identify which byproducts affect growth and how
  2. Team 2 - Modify C. hutch
    1. Build shuttle vector
      1. plasmids require ori, promoter, genes, restriction sites, selection marker genes
      2. a shuttle vector has everything it need to replicate both in E. coli and C. hutch (need C. hutch promoter, C. hutch ori, C. hutch 
      3. Come up with a map, build a cloning strategy to get there based on the paper
      4. Design, order DNA, and start building (should get done a week before June 8th since the order takes about a week)
  3. Team 3 - modify E. Coli
    1. cloning workflow
    2. Circuit design
      1. Modeling (metabolic models, ODEs of growth rates, etc.)
      2. Sequences to put in E. Coli
        1. LuxR, communication
        2. FAEE pathway + sequences (which ones)
    3. Measuring FAEEs - concrete plan
      1. The peaks from the chromatography method may not be distinguishable
      2. extract from cells?
      3. assay development
    4. Measure byproducts that may affect growth
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