Team:Harvard/Dailybook/Week7/Widgetry

From 2008.igem.org

Contents

Goals for this week

  • Shewanella and E. coli co-culture experiments
    • If Shewanella responds rapidly to lactate, and E. coli breaks down lactose into lactate, can we couple lactose breakdown by E. coli with current production by Shewanella?
    • Motivation:
      • An inducible system in E. coli could be used to control Shewanella current production

Monday: August 4, 2008

Co-culture experiment setup

  • Chambers 1 & 2: wt E. coli + wt Shewie + lactose
  • Chambers 4 &5 : Lac-operon knockout E. coli + wt Shewie + lactose

Positive Controls

  • Chamber 3: wt E. coli + wt Shewie + lactate
  • Chamber 6: Lac-operon knockout E. coli + wt Shewie + lactate

Negative Controls

  • Chamber 7: wt Shewie + lactose
  • Chamber 8: wt E. coli + lactose
  • Chamber 9: Lac-operon knockout E. coli + lactose

Tuesday: August 5, 2008

Co-culture experiment results, day 1

Co-culture part1.JPG
  • Notes:
    • At t = 2000 s, all cells added
    • At t = 80000 s, all injection done


Zoomed-in graph of results, part 1

Co-culture part1 zoomed.jpg
  • Notes:
    • Zoomed-in on time since injections
    • Positive controls omitted


Preliminary Conclusions

  • Noticeable difference in behavior between wt E. coli + Shewie and Lac-operon knockout + Shewie co-cultures
  • Positive and negative controls show most likely no other variables involved
  • Could be used to create tic-tac-toe which uses the difference in behavior to determine moves

Wednesday: August 6, 2008 to Friday: August 8, 2008

Co-culture experiment results, day 1 & 2

wt E. coli vs. Lac-operon knockout E. coli

Co-culture part2 test.jpg
  • Notes:
    • At t = 260000 s, adjusted gas for Chamber 5
    • At t = 340000 s, lactose injection for Chamber 1
    • Initially, very noticeable difference between wt E. coli and Lac-operon knockout E. coli, but the current production of one of the E. coli chambers rises after a day, making the difference less distinguishable

Results w/ positive controls

Co-culture part2 wpos.jpg
  • Notes:
  • At t = 340000 s, lactose injections for Chambers 1, 3, and 6
    • Wanted to see if the positive controls would exhibit the same behavior when lactose injected instead of lactate


Results (entire)

Co-culture part2.jpg
Concerns
  • Chambers with co-cultures contained double the amount of cells of the controls
  • Current production in non-positive co-culture chambers
    • wt Shewie + lactose
    • lac-operon knockout E. coli + Shewie + lactose
    • lac-operon knockout E. coli + lactose
Conclusions
  • Although behavior of wt E. coli + Shewie and Lac-operon knockout E. coli + Shewie co-cultures are not as distinguishable after a day, our focus can only be on the first day
  • Will try the same experiment with DH5(alpha) and DH5(alpha)/pUC19 strains
  • Need to look for inducible lacZ systems
    • Light-repressible (RU1012 with plasmid) system?
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