Difference between revisions of "Part:BBa K1421006:Experience"
 
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Our team helped increase characterization of the part Bba_K1421006(RpaI). 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_K1421006(RpaI). 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.  
  
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As shown below, RpaI was able to induce F2620 in this visual induction, as colonies in the top right section did produce GFP. This is not the expected result, since the Canton Lab showed that the Rpa AHL (p-Coumaroyl-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, RpaI was able to induce F2620 in this visual induction, as colonies in the top right section did produce GFP. These results indicate that RpaI will crosstalk with LuxR and F2620.   
 
<div style="text-align: center;">[[File:T--Arizona_State--rpaplate.png]]</div>
 
<div style="text-align: center;">[[File:T--Arizona_State--rpaplate.png]]</div>
 
<div style="text-align: center;">Plate with GFP+(top left), Sender(center), -Receiver(bottom) and F2620(top right)</div>  
 
<div style="text-align: center;">Plate with GFP+(top left), Sender(center), -Receiver(bottom) and F2620(top right)</div>  
  
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The figure below compares RpaI at 10% and 50% concentrations alongside the native AHL system LuxI at 10% and 50% concentrations. RpaI 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.  
+
The figure below compares RpaI at 10% and 50% concentrations alongside the native AHL system LuxI at 10% and 50% concentrations. RpaI is shown to induce F2620, but to a lesser degree than LuxI. This affirms that F2620 is capable of being induced by RpaI synthesized within BL21(DE3) E. coli, supporting the notion that crosstalk is occurring. This result corroborates the plate induction result, indicating that p-Coumaroyl AHL will induce F2620.
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<div style="text-align: center;">[[File:T--Arizona_State--lasind.png]]</div>
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<div style="text-align: center;">[[File:T--Arizona_State--rpaind.png]]</div>
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<div style="text-align: center;">GFP absorbance from LasI over time</div>
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<div style="text-align: center;">GFP absorbance from RpaI over time</div>
  
 
====AHL Disposal Test====
 
====AHL Disposal Test====
  
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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 LasI produces a 3-oxo AHL, which should have been destroyed by bleach.  
+
The final experiment conducted using this part aimed to determine proper safe disposal procedures for the p-Coumaroyl 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 RpaI were NOT properly deactivated by a 10% bleach solution. This was the expected result, as RpaI does not produce a 3-oxo AHL, which should not have been destroyed by bleach.  
−
<div style="text-align: center;">[[File:T--Arizona_State--lasbleachgraph1.png]]</div>
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<div style="text-align: center;">[[File:T--Arizona_State--rpableachgraph.png]]</div>
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<div style="text-align: center;">GFP absorbance from LasI over time</div>
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<div style="text-align: center;">GFP absorbance from RpaI over time</div>
  
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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.
+
A standard 15 minute Liquid autoclave cycle was also used to treat an extracted AHL solution. The figure below indicates that RpaI 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--lasautoclavegraph1.png]]</div>
+
<div style="text-align: center;">[[File:T--Arizona State--rpaautoclavegraph1.png]]</div>
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<div style="text-align: center;">GFP absorbance from LasI over time</div>
+
<div style="text-align: center;">GFP absorbance from RpaI over time</div>
  
 
====Conclusion====
 
====Conclusion====
  
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The results demonstrate that Las was able to effectively induce F2620 after being extracted. The Las results were consistent, which showed significantly decreased induction when treated with bleach, indicating 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, for LasI, both bleach and autoclaving are capable of deactivating 3-oxo-C12-HSL.  
+
The results demonstrate that RpaI was able to effectively induce F2620 after being extracted. The Rpa results were inconsistent, which showed varying levels of induction when treated with bleach, with no indication 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, for RpaI, bleach is unable to effectively degrade the Rpa AHL but autoclaving is sufficient.
 
===User Reviews===
 
===User Reviews===
 
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Latest revision as of 05:27, 24 October 2016


This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K1421006

Characterization of BBa_K1421006-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_K1421006(RpaI). 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, RpaI was able to induce F2620 in this visual induction, as colonies in the top right section did produce GFP. These results indicate that RpaI will crosstalk with LuxR and F2620.

T--Arizona State--rpaplate.png
Plate with GFP+(top left), Sender(center), -Receiver(bottom) and F2620(top right)

The figure below compares RpaI at 10% and 50% concentrations alongside the native AHL system LuxI at 10% and 50% concentrations. RpaI is shown to induce F2620, but to a lesser degree than LuxI. This affirms that F2620 is capable of being induced by RpaI synthesized within BL21(DE3) E. coli, supporting the notion that crosstalk is occurring. This result corroborates the plate induction result, indicating that p-Coumaroyl AHL will induce F2620.

T--Arizona State--rpaind.png
GFP absorbance from RpaI over time

AHL Disposal Test

The final experiment conducted using this part aimed to determine proper safe disposal procedures for the p-Coumaroyl 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 RpaI were NOT properly deactivated by a 10% bleach solution. This was the expected result, as RpaI does not produce a 3-oxo AHL, which should not have been destroyed by bleach.

T--Arizona State--rpableachgraph.png
GFP absorbance from RpaI over time

A standard 15 minute Liquid autoclave cycle was also used to treat an extracted AHL solution. The figure below indicates that RpaI was nearly completely destroyed via autoclaving. This was the expected result, as the high pressure and temperatures should deactivate any AHL molecules present.

T--Arizona State--rpaautoclavegraph1.png
GFP absorbance from RpaI over time

Conclusion

The results demonstrate that RpaI was able to effectively induce F2620 after being extracted. The Rpa results were inconsistent, which showed varying levels of induction when treated with bleach, with no indication 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, for RpaI, bleach is unable to effectively degrade the Rpa AHL but autoclaving is sufficient.

User Reviews

UNIQad3720c520239736-partinfo-00000000-QINU UNIQad3720c520239736-partinfo-00000001-QINU