Part:BBa_C0170:Experience

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Characterization of BBa_C0170-Arizona_State 2016

Authors: Ernesto Luna, Brady Dennison, Cassandra Barrett, Jimmy Xu, Jiaqi Wu, Dr. Karmella Haynes

Our team helped increase characterization of the part Bba_C0170(RhlI). This part was tested against its ability to induce the part BBa_F2620 by the Canton Lab(MIT). This part outputs PoPS as a Receiver Device combined with LuxR. An induction test on BBa_F2620 had been done by Dr. Barry Canton (2008), but they tested GFP production over various AHL concentrations, while our test was an 8-hour GFP read over time for 2 AHL concentrations (10 and 50%). In addition, the Canton test utilized synthetic AHLs while our test utilized AHLs produced via an E.coli chassis. A visual induction test was also done, plating the Sender alongside a GFP positive control, negative receiver control, and F2620.

As shown below, Rhl was able to induce F2620 in this visual induction, as colonies in the top right section did produce GFP. This was a lower level of induction, so although crosstalk may occur, it is minimal. This is also affirmed by the Canton data, which showed that C4-HSL is able to induce F2620, but only at higher concentrations.

Plate with GFP+(top left), Sender(center), -Receiver(bottom) and F2620(top right)

The figure below compares RhlI at 10% and 50% concentrations alongside the native AHL system LuxI at 10% and 50% concentrations. RhlI is shown to induce F2620, but to a much lesser degree than LuxI. This affirms that F2620 is capable of being induced by RhlI synthesized within BL21(DE3) E. coli, supporting the notion that crosstalk is occurring. This result is supported by the plate induction result, as both indicate that a low degree of crosstalk will occur between F2620 and RhlI.

GFP absorbance from RhlI over time

Conclusion

The results demonstrate that RhlI was able to induce F2620 after being extracted to a minimal degree. These results were consistent across both the visual plate induction and the 8-hour plate read. Because RhlI is capable of inducing F2620, the Rhl and Lux systems cannot be characterized as orthogonal systems, which may limit its use in any constructed genetic circuits.

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