Team:UNIPV-Pavia/New Parts

Conclusions
We demonstrated that this part works as expected, sensing 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, 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 for both LB and M9).

This part shows to have a very low leakage rate (about 0.025 RPU) but also a very low transcription rate for high concentrations of aTc. So, it can be used for tight regulation, in those systems who need very low leakage rates (no gene expressed in absence of inducer) but not for massive protein production.

Unfortunately, the estimated transfer function is very noisy (probably because the measured fluorescence values are very close to blank values), and further experiments should be performed in order to validate these induction curves.

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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.



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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Description
This should work as a lactose/IPTG sensor.

BBa_J23118 promoter drives the constitutive production of lacI repressor ( BBa_C0012 ), which inhibits lac promoter ( BBa_R0011 ) activity. When lactose or IPTG is added to the medium, it binds lacI and inhibits it. So, the PoPS output is a function of lactose/IPTG concentration.

Thanks to the hybrid lac promoter ( BBa_R0011 ), designed taking the Plambda promoter ( BBa_R0051 ) and substituting its cI ( BBa_C0051 ) binding sites with two lacI binding sites, the behaviour of this device is not a function of glucose concentration because the wild type CAP binding sites are not present in this artificial lac promoter.

The test performed to characterize this device has been done with BBa_K173012, which is the measurement system of BBa_K173010 itself.



Characterization

 * We inoculated 8 ul of BBa_K173012 glycerol stock in 5 ml of LB + Amp and incubated it overnight (37°C, 220 rpm).
 * In the morning we diluted 1:100 the culture in two falcon tubes and incubated them under the same conditions as before.
 * After about 6 hours we added IPTG in one of the two cultures (final concentration = 1 mM).
 * Fluorescence and O.D.600 were measured in the microplate reader after additional 4 hours.

Results: both cultures had a comparable O.D.600, but also had the same RFU values, demonstrating that the induced culture did not produce any GFP.

Conclusions
We did not perform any standard measurement on this device because preliminary tests showed that GFP levels of induced and non induced cultures were the same and were equals to BBa_B0033 negative control.

Unfortunately, we did not check if the sequence of BBa_K173012, so we do not know if it was actually correct. Another possibility is that lacI is produced at so high levels that 2 mM of IPTG is not sufficient to induce the lac promoter.

Further tests should be done for this system. Top