Each one of the input, processor and output inserts [dp003] [dp011] and [dp013] selected prior have been cloning using cloning gate technique into all three of the pDuet vectors [pACY],[pCDF] and [pColA] . [golden gate protocol].Following golden gate, we find ourselves with [pACY-dp003] [pCDF- dp011] and [pColA-dp013] plasmid constructs, each one is transformed into E. coli of the DH5α strain.

Figure 1. shows the circuit's difference in fluorescence over OD at varying concentrations of pyocyanin. We can see that our device successfully transmits the signal received in input as readable output. We can also see that the communication between different parts of our System is perfectly correlated to the concentration of pyocyanin that we give as an input, meaning that our device successfully transmits the signal received in input as a readable output depending of the activity of the input.

The Fluorescence/OD is reaching ~45000 for an added 20 μM pyocyanin concentration. This data shows that pyocyanin at 20 μM is the optimal concentration (yielding the highest fluorescence/ OD), after this concentration, cells begging to die because of the toxicity of pyocyanin, so for further experiences, we recommend a 13 uM working concentration.

Figure 2. This graph shows the circuit's response, measured in fluorescence/ OD, to varying concentrations of OC6. The combination of the three inducers piocynin , OHC14 and OC6 has been added to the genetic circuit composed of that is composed of an input, a processor and output. The concentration of OC6 varies from 0.1 to 1000 μM but the concentrations of OHC14 and piocynin remain at, respectively 0.1 μM and 10 μM throughout.

This graph shows that the varying concentration of OC6 ranging from 0.1 to 1000 μM affects the circuit's Fluorescence/OD. The fluorescence/OD for an OC6 concentration of 1000 μM is at 6000 while the fluorescence/OD for an OC6 concentration at 62.5 is at 220000. This data proves that OC6 which induces the processor promoter has a major effect on protein translation.

Figure 3. This graph shows the circuit's response, measured in fluorescence/ OD, to varying concentrations of OCH14. The combination of the three inducers piocynin , OHC14 and OC6 has been added to the genetic circuit composed of that is composed of an input, a processor and output. The concentration of OCH14 varies from 0.015 to 1000 μM but the concentrations of OC6 and piocynin remain constant at, respectively 0.1 μM and 10 μM throughout.

This graph shows that the varying concentration of OCH14 ranging from 0.05 to 1000 μM affects the circuit's Fluorescence/OD. The fluorescence/OD for an OCH14 concentration of 0.1 μM is at 22500 while the fluorescence/OD for an OCH14 concentration at 250 μM is at 19000. This data proves that OCH14 which induces the processor promoter has a major effect on protein translation and that the optimal concentration is 0.1 μM.

These 3 graphs shows that varying concentrations of piocynin, OHC14 and OC6 each individually have an effect on circuit fluorescence/OD. The variation in circuit fluorescence/ OD reflects on the amount of fluorescent protein translated. Hence, each of the chemical inducers, have an impact as to the amount to fluorescent protein translated and has been proven to have direct effect on the overall genetic circuitry.

Figure 4. This bar plot regroups data from figures 1, 2 and 3 and displays the maximum fluorescence/OD of the circuit upon activation by different inducers at different concentrations.

This data shows that, without any added inducers, the maximum fluorescence/OD reach is around 9000. In the presence of chemical inducers, the fluorescence/OD can vary from 20000 to up to 50000 which is significantly higher than the maximum value reach without any inducers added. Hence, the circuitry needs to induced by chemical inducers to general a intensity and readable output signal.

It is also observed that the highest maximum fluorescence/OD of 50000 reached is when the system is induced only by piocynin at 20 μM. In comparison, the fluorescence/OD reached when all three inducers added, at all concentrations is significantly small.

However, the higher quantify of piocynin (20 uM vs 10 uM in others) is not the only cause of this higher fluorescence/OD as the system induced by piocynin 30µM+ 0.1 µM OC6+ 0.01 µM OHC14 reached a value of 28000 vs. 38000 for induction piocynin 10µM+ 15.6 µM OC6+ 0.01 µM OHC14. This data shows that the system induces a different intensity of output signal in accordance to the different concentrations of chemical inducers added.

Figure 5. This graph shows that the circuit's on which piocynin only has been added has a higher fluorescence/OD than does the circuit where piocynin, OCH14 and OC6 has been added. This signifies that the system has an overall higher Red fluorescent protein rate when pyocinin is added only vs when the triade of chemical inducers piocynin, OCH14 and OC6 are added. This finding leads to the conclusion that adding OCH14 and OC6 to the system creates a resistance to the output signal.