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Experiments

• Open Loop [Cc]

i) Cells were transferred to 50 mL Glu M9 in 250 mL flask with 5 ng/mL [aTc] and 1,0.1 uL/mL of inoculum.

ii) K12z1 was inoculated in 5 mL Glu M9 in 50 mL tube with [aTc]=5 ng/mL and 0.5,0.05 uL/mL of inoculum.

iii) However, right OD was not reached in time and this would be repeated.

• Open Loop [Dd]

i)Cells were transferred to 50 mL Glu M9 in 250 mL flask with [aTc]=1 ng/mL and inoculum=1,0.1 uL/mL.

ii) K12z1 was inoculated in 5 mL Glu M9 in 50 mL tube with [aTc]=1 ng/mL and inoculum=0.5,0.05 uL/mL.

iii)The flask having cells with OD=0.193 was filtered out.

• 7 dilutions with full IPTG conc range were made for pL.LuxR.Y.pR.C with AI from 1 ng/mL aTc and new 2xM9 in 1:1 ratio.
  • -ve control: K12z1 in AI from 1 ng/mL aTc and new 2xM9 in 1:1 ratio
  • +ve control: pL.Y, 100 uM IPTG, 1x M9

• To show that K12z1 does not produce any chemical in M9+aTc which might affect the system, we compared the CFP/YFP expressions of the following 2 samples:
  • pL.LuxR.Y.pR.C was inoculated in Glu M9 with 500 uM IPTG, and
  • K12Z1 was grown in 1 ng/mL aTc and the cells were filtered out. This medium was then diluted with 2xGlu M9 in a 1:1 ratio, and pL.LuxR.Y.pR.C was inoculated in it with 500 uM IPTG.

• pL.LuxR.Y.pR.C, K12z1, pL.Y were Inoculated in LB

• pL.LuxR.Y.pR.C and K12z1 were inoculated in LB.

• pT.LuxI.C, K12Z1 were inoculated in LB for open loop duplicate expts [Ee] and [Cc].

Microscopy

• Open loop samples [Aa] were imaged.

• Open loop samples [Ff] were imaged.

Analysis