Part:BBa_K173011:Experience

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BBa_K173011 - aTc inducible device with J23118 promoter - UNIPV-Pavia (Test performed by)

Description

This is an aTc sensing device.

BBa_J23118 promoter drives the constitutive production of tetR repressor (BBa_C0040), which inhibits tetR promoter (BBa_R0040) activity. When aTc is added to the medium, it binds tetR and inhibits it. So, the PoPS output is a function of the aTc concentration.

A less tight regulation is expected for this inducible system than in BBa_K173007 because BBa_J23118 promoter is weaker than BBa_J23100 and so tetR repressor shold be produced at lower levels than in the other sensor.

The data below are referred to BBa_K173026, which is the measurement system of BBa_K173011.

Characterization

aTc concentration [ng/ml]	LB		M9 supplemented
	Doubling time [minutes]	RPU	Doubling time [minutes]	RPU
0	36	0.51 [ 0.43 ; 0.73]	78	0.96 [ 0.80 ; 1.23]
25	33	1.36 [ 1.28 ; 1.44]	82	1.66 [ 1.38 ; 2.11]
50	40	1.39 [ 1.30 ; 1.48]	81	1.85 [ 1.61 ; 2.27]
75	43	1.25 [ 1.17 ; 1.31]	88	1.75 [ 1.51 ; 2.15]
100	43	1.40 [ 1.28 ; 1.48]	90	1.89 [ 1.68 ; 2.34]
200	45	1.37 [ 1.31 ; 1.41]	92	1.75 [ 1.51 ; 2.09]
300	48	1.45 [ 1.36 ; 1.55]	94	1.73 [ 1.53 ; 2.07]

BBa_K173026 Growth curves for BBa_K173026 in M9

BBa_K173026 Growth curves for BBa_K173026 in LB

BBa_K173026(dGFP/dt)/OD in LB and M9

BBa_K173026(dGFP/dt)/OD in LB and M9

BBa_K173026 Induction curve of BBa_K173026 in M9

BBa_K173026 Induction curve of BBa_K173026 in LB

Conclusions

We demonstrated that this part works as expected because GFP is produced as an increasing function of the aTc concentration provided in the culture medium. The transfer function of this sensor has been characterized in standard units (RPUs) in two different growth media (LB and M9 supplemented with glycerol), as well as the metabolic burden (in terms of doubling time) which affects E. coli bearing this part.

On the other hand, as for BBa_K173007, we did not expect to have a higher GFP synthesis rate per cell after the exponential growth phase than in the exponential phase itself (as reported in the 3rd plot).

This part shows a high level of leakage (about 0.5 RPU in LB and 1 RPU in M9) but also high levels of induction for high concentrations of aTc (about 1.45 RPU in LB and 1.7 RPU in M9). For this reason, it can be used in those cases where an important response in terms of gene expression is required in presence of an inducer, but is not suitabe if the absence of expression has to be off when inducer is absent. Its induction range is 0.95 RPU in LB and 0.7 RPU in M9.

The switch point of this device is between 0 ng/ml and 25 ng/ml of aTc in both LB and M9.

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