Difference between revisions of "Part:BBa C0170:Experience"

Latest revision as of 05:19, 24 October 2016

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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(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.

User Reviews

UNIQe610b56fdd207e3a-partinfo-00000000-QINU

Antiquity

This review comes from the old result system and indicates that this part did not work in some test.

UNIQe610b56fdd207e3a-partinfo-00000002-QINU

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 ===Applications of BBa_C0170===
-===Characterization of Bba_C0161-Arizona_State 2016===
+===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.
+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, 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.
+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.
-<div style="text-align: center;">[[File:T--Arizona_State--lasplate.png]]</div>
+<div style="text-align: center;">[[File:T--Arizona_State--rhlplate.png]]</div>
 <div style="text-align: center;">Plate with GFP+(top left), Sender(center), -Receiver(bottom) and F2620(top right)</div>
-The figure below compares LasI at 10% and 50% concentrations. LasI is shown to induce F2620. This affirms that F2620 is capable of being induced by LasI synthesized within BL21(DE3) E. coli. 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.
+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.
-<div style="text-align: center;">[[File:T--Arizona_State--lasind.png]]</div>
+<div style="text-align: center;">[[File:T--Arizona_State--rhlind.png]]</div>
-<div style="text-align: center;">GFP absorbance from LasI over time</div>
+<div style="text-align: center;">GFP absorbance from RhlI over time</div>
-====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.
-<div style="text-align: center;">[[File:T--Arizona_State--lasbleachgraph1.png]]</div>
-<div style="text-align: center;">GFP absorbance from LasI over time</div>
-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.
-<div style="text-align: center;">[[File:T--Arizona State--luxautoclavegraph.png]]</div>
-<div style="text-align: center;">GFP absorbance from LuxI over time</div>
 ====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.
+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.
 ===User Reviews===