Part:BBa_K2033014
p-Coumaroyl HSL Sender-RpaI
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 43
- 1000COMPATIBLE WITH RFC[1000]
Short Description
This is a synthase enzyme that produces p-Coumaroyl HSL. This AHL synthase is designed to be inserted into a modular sender vector BBa_K2033011 with a constitutive Tet promoter, 2 ribosome binding sites (RBSs), an RFC10 prefix and mCherry.
Introduction to AHL Quorum Sensing
AHL quorum sensing functions within two modules. The first module, the "Sender," must be induced by certain environmental conditions, usually population density of surrounding organisms. This will begin production of the AHL by an AHL synthase, which is then detected by the second module, the "Receiver." The Receiver will cause the expression or silencing of certain genes to achieve the desired purpose of the communication, whether it is the production of GFP or to increase growth rate.
The Rpa system is from the bacteria Rhodopseudomonas palustris. The Rpa system produces a unique AHL molecule, which is shown below:
This AHL possesses a coumaroyl tail, which is the primary recognition factor for RpaR.
Characterization of BBa_K1421006-Arizona_State 2016
Authors: Ernesto Luna, Brady Dennison, Cassandra Barrett, Jimmy Xu, Jiaqi Wu, Dr. Karmella Haynes
After gel verification and sequencing, the RpaI part was retransformed in BL21(DE3) E. coli and run in a 96-well plate from 580-610nm to measure mCherry production. This produced the curve below, suggesting that the AHL is being produced by the sender, since mCherry production increased over an 8-hour read time. The mCherry gene lies downstream of the RpaI synthase gene, so mCherry production is a good indicator of Rpa AHL production.
Gas chromatography was also done on the E. coli cultures to confirm production of the AHL molecule by the E. coli chassis. These tests are still in progress and will be completed at a later date.
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.
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.
Safety
This section aims to provide safety information and suggestions about the AubI part. The greatest concern from this part is the activation of pathogens via crosstalk. According to Integrated Device Technologies, quorum sensing genes are not considered dangerous by themselves, as they do not directly cause the creation of a new pathogenic strain. They may contribute to pathogenicity, but so do synthetic promoters. So, the actual AHL molecules are the chief concern.
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.
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.
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.
References
(1)Hirakawa1, Hidetada, Oda1, Yasuhiro, Phattarasukol, Somsak. "Activity of the Rhodopseudomonas palustris p-Coumaroyl-Homoserine Lactone-Responsive Transcription Factor RpaR." J. Bacteriol. 193.10. (2011) 2598-2607.
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