OBJECTIVE
Determine the population density at which the quorum system gets activated and provide the modellers with data to infer a_LuxI, the production rate of LuxI.

PROCEDURE
We transformed E. Coli with a regulator and an actuator plasmid, containing constitutive LuxR and Plux, sfGFP, mCherry and LuxI respectively (Figure 2).

Subsequently, we let these colonies grow to different final population densities. This was achieved by varying glucose concentrations in a defined medium. [1] Population density was assessed by measuring absorbance at 600 nm wavelength. Fluorescence emitted by sfGFP and mCherry served as a read-out of the level of activation. A detailed protocol is available in Protocols.

RESULTS

CONCLUSION

• We can modulate the density a bacterial population reaches in defined medium by varying the amount of glucose.
• The quorum sensing system shows a response to increasing population densities.

OBJECTIVE
Determine expression levels of GFP production under the control of the AND-gate with different inducer concentrations. In this experiment we wanted to assess whether our designs would be capable to distinguish healthy and tumor tissue based on lactate and expected AHL concentrations.

PROCEDURE
Two plasmids required for the AND-gate were transformed into E. coli Top 10 (Figure 4). Cultures were grown in microtiter plates under combinations of 8 different AHL and 8 different lactate concentrations and measured after 5.5 hours. A detailed protocol is available in Protocols.

RESULTS
The different conditions cleary have an impact on expression levels of sfGFP under control of the AND-gate promoter. All three designs show increasing activation with increasing inducer concentration, even if the second inducer is not present. The highest fold-change for all designs however, is observed if both inducers are present in high amounts.

CONCLUSION

• Leakiness of the synthetic promoter increases with increasing amounts of either inducer in the absence of the other.
• Increasing AHL amounts have a greater influence on the leakiness in absence of lactate.
• All three AND-gates exhibit highest inductions in presence of both inducers.
• At lactate levels found in healthy tissue and low AHL concentrations, all designs are only weakly activated.
• Design B performed best at distinguishing “healthy tissue lactate”, low AHL vs. “tumor tissue lactate”, high AHL. Design C, on the other hand, performed worst.

OBJECTIVE
Verify the findings of the AND-gate characterization without quorum sensing with strains of E. Coli that contain additionally to the AND-gate also LuxI, the enzyme that catalzyes AHL production.

PROCEDURE
Two plasmids required for the AND-gate were transformed into E. coli Top 10 (Figure 6). Cultures were grown over night in deep-well plates in media with varying lactate concentrations. The measurements of population density and GFP fluorescence were taken after ~16 hours on a plate reader. A detailed protocol is available under Protocols.

RESULTS
The data is very noisy and it’s hard to make general statements about this systems behaviour. Despite this, a clear trend is visible for GFP to be higher expressed under lactate concentrations similar to tumor tissue than under those resembling healthy tissue or no lactate at all. With increasing population densities this effect becomes less pronounced (Figure 7).

CONCLUSION

• Due to a lot of noise in the data, conclusions have to be drawn with caution
• Under lactate concentration mimicking tumor tissue, GFP gets stronger expressed than under lactate levels associated with healthy tissue.
• Fold-changes are around 4 for design B and 2 for design A which is considerably less than observed in Figure 5. This might be due to a somewhat different experimental setup (see Protocols) that lead to accumulation of GFP. Another explanation could be that the amounts of AHL are lower than the ones used in the experiment "AND-gate without Quorum Sensing".