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Experiments

• Open loop experiment [E]

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

ii) pL.LuxR.Y.pR.C and K12Z1 were inoculated in LB.

iii) K12z1 was inoculated in Glu M9 and atc (1000)

iv) The contents of the flask with required OD (50 ng/mL) were filtered.

v) Seven dilutions for pL.LuxR.Y.pR.C were made with 50 old:50 new 2xM9 along with -ve control: K12z1

• The following -ve controls were also done :

i) pL.LuxR.Y.pR.C with K12Z1 grown medium,500 uM IPTG.

ii) pL.LuxR.Y.pR.C with new M9,500 uM IPTG.

• Open loop experiment [F]

i) Cells were transferred to 50 mL Glu M9 in 250 mL flask with [aTc]=0 ng/mL and inoculum=1,0.1 uL/mL

ii) pL.LuxR.Y.pR.C, K12Z1 and pL.YFP were inoculated in LB

iii) The contents of the flask with required OD ( ) were filtered.

Microscopy

• pT.LuxI.C imaging was done as part of the equivalence expt.

• pL.LuxR.Y imaging was done as part of the eqiuvalence expt.

Analysis

• We obtained the following scatter plots (mean log) for the equivalence curves:

pL.luxR.YFP	pT.luxI.CFP
mean CFP vs IPTG conc.	mean CFP vs IPTG conc.
mean YFP vs IPTG conc.	mean YFP vs IPTG conc.
Scatter plot: Each dot here represents the fluorescence value of an individual cell. The Cyan dots represent the fluorescence values of wild-strain K12z1 cells.

Inference