Team:Chiba/Demo experiments:Senders

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Demo Experiment ~Senders~

Method

  1. Pre-culture
    1. Picked and cultured the following glycerol stocks in 2mL of LB:
      1. LB-Amp, BBa_T9002, (JW1908)
      2. LB-Amp+0.2%Glu, BBa_K084012(plac+rbs+LuxI(no LVA)), (XL10G)
      3. LB-Amp+0.2%Glu, BBa_K084007(plac+rbs+LasI(no LVA)), (XL10G)
    2. Cultured at 37°C for 12h.
  2. Culture
    1. Added 6.25% each of the pre-cultures to new LB medium.
      1. LB-Amp, BBa_T9002
      2. LB-Amp+0.2%Glu, BBa_K084012(plac+rbs+LuxI(no LVA)), BBa_K084007(plac+rbs+LasI(no LVA))
    2. Cultured at 37°C for 4~5h
  3. Wash
    1. Transfer 10mL each of the culture to 50mL centrifuge tubes.
    2. Centrifuged for 6min at 3600rpm,20°C and discarded the supernatant.
    3. Added LB-Amp to each centrifuge tube:
      1. 10mL to the tube that contains BBa_T9002
      2. 5mL to the tube that contains BBa_K084012, BBa_K084007
    4. Centrifuged for 6min, 3600rpm at 20°C the tube containing BBa_K084012, BBa_K084007 and discarded the supernatant.
    5. 10mL to the tube that contains BBa_K084012, BBa_K084007
    6. Centrifuged for 6min, 3600rpm at 20°C the tube containing BBa_K084012, BBa_K084007 and discarded the supernatant.
    7. 5mL to the tube that contains BBa_K084012, BBa_K084007
  4. Mix
    1. Mixed the sender cells BBa_K084012 and BBa_K084007 both with BBa_T9002 at a 1:1 ratio.
    2. Added 100μL each to a 96-well shallow plate (as shown in the figure).
      1. Green part is BBa_K084012:BBa_T9002=1:1
      2. Red part is BBa_K084007:BBa_T9002=1:1
      3. Uncolored part is BBa_T9002 alone.
  5. Culture and observe results

Results

Green region: sender=LuxI, Red circular region: sender=Las I.


LuxI GFP is detected at 4h following mixing while LasI GFP is detected after 8h, thus successfully demonstrating time-delay depending on the sender used.

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