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Part:BBa_C0170:Experience
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Applications of BBa_C0170
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(LasI). 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, Las was unable to induce F2620 in this visual induction, as colonies in the top right section did not produce GFP. This is not the expected result, since the Canton Lab showed that the Las AHL (3-oxo-C12-HSL) was capable of inducing F2620. This may have been due to an issue with AHL diffusion on the plate and will be examined more in the plate reader test.
The figure below compares LasI at 10% and 50% concentrations alongside the native AHL system LuxI at 10% and 50% concentrations. LasI is shown to induce F2620, but to a lesser degree than LuxI. This affirms that F2620 is capable of being induced by LasI synthesized within BL21(DE3) E. coli, supporting the notion that crosstalk is occurring. This result contrasts with the plate induction result, but because it is supported by the Canton results, it is likely that the plate induction for LasI was erroneous.
AHL Disposal Test
The final experiment conducted using this part aimed to determine proper safe disposal procedures for the 3-O-C12-HSL. This AHL molecule is capable of crosstalk with potentially pathogenic strains of bacteria, and proper disposal of these AHLs should be an important biosafety measure taken. S.A. Borchardt had already tested the susceptibility of AHLs to bleach and found that 3-oxo AHLs were easily broken down by bleach while other AHLs were not. Our experiment aimed to test the application of standard EH&S sanitation protocols on AHLs (10% bleach solution and autoclaving). The figure below indicates that AHLs produced by LasI were properly deactivated by a 10% bleach solution. This was the expected result, as LuxI produces a 3-oxo AHL, which should have been destroyed by bleach.
A standard 15 minute Liquid autoclave cycle was also used to treat an extracted AHL solution. The figure below indicates that LasI was nearly completely destroyed via autoclaving. This was the expected result, as the high pressure and temperatures should deactivate any AHL molecules present.
Conclusion
The results demonstrate that Lux was able to effectively induce F2620 after being extracted. The Lux results were not consistent, which showed decreased induction when treated with bleach, but no evidence of complete AHL inactivation. According to the autoclave results, a standard 15 min liquid procedure is able to degrade nearly all AHLs. The extreme pressure and temperature generated by the autoclave was more than enough to remove any threat posed by these AHL samples. In summary, our data suggests that autoclaving will ensure at least some inactivation of AHLs, while bleach may have no effect at all on the degradation of AHLs.
User Reviews
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