Part:BBa_K2033012
3-oxo-C6-HSL Sender- EsaI
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 273
Illegal NgoMIV site found at 528
Illegal AgeI site found at 580 - 1000COMPATIBLE WITH RFC[1000]
Short Description
This is a synthase enzyme that produces 3-oxo-N-[(3S)-2-oxooxolan-3-yl]hexanamide (3-O-C6-HSL). This AHL synthase is designed to be inserted into a modular sender vector BBa_K2033012 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 Esa system was discovered to be from the organism Erwinia stewartii. The Esa system produces an AHL molecule, which is shown below:
This AHL possesses an 3-oxo acyl tail, which is the primary recognition factor for EsaR. The further characterization of this part is shown in the Design portion of this part.
Characterization of BBa_K1670004-Arizona_State 2016
Authors: Ernesto Luna, Brady Dennison, Cassandra Barrett, Jimmy Xu, Jiaqi Wu, Dr. Karmella Haynes
After gel verification and sequencing, the EsaI 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 EsaI synthase gene, so mCherry production is a good indicator of Esa 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_K1670004(EsaI). 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, Esa was able to strongly induce F2620 in this visual induction, as colonies in the top right section did produce GFP. This is the expected result, as Esa produces the same AHL as Lux (3-oxo-C6-HSL) and the Canton Lab showed that the Lux AHL was capable of inducing F2620.
The figure below compares EsaI at 10% and 50% concentrations alongside the native AHL system LuxI at 10% and 50% concentrations. EsaI is shown to induce F2620 to an even greater degree than LuxI. This may be due to the fact that E. coli has machinery that is able to more efficiently produce the AHL using the Esa synthase. This affirms that F2620 is capable of being induced by EsaI synthesized within BL21(DE3) E. coli, supporting the notion that crosstalk is occurring. This result corroborates the plate induction result.
AHL Disposal Test
The final experiment conducted using this part aimed to determine proper safe disposal procedures for the 3-O-C16-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 autoclaving on 3-O-C6-HSL, a standard EH&S sanitation protocol. A standard 15 minute Liquid autoclave cycle was used to treat an extracted AHL solution. The figure below indicates that Esa was not completely destroyed via autoclaving. This was NOT the expected result, as the high pressure and temperatures should have deactivated any AHL molecules present.
Conclusion
The results demonstrate that Esa was able to effectively induce F2620 after being extracted. The Esa disposal results were inconsistent, which showed decreased induction when autoclaved, but was not capable of completely destroying the AHLs. The extreme pressure and temperature generated by the autoclave should have been more than enough to remove any threat posed by these AHL samples. This may have been an experimental error, and should be evaluated further in future experiments.
References
(1)Davis, René Michele, Ryan Yue Muller, and Karmella Ann Haynes. "Can the Natural Diversity of Quorum-Sensing Advance Synthetic Biology?" Frontiers in Bioengineering and Biotechnology 30th ser. 3 (2015)
(2) von Bodman, S Beck, Farrand, S.K. "Capsular polysaccharide biosynthesis and pathogenicity in Erwinia stewartii require induction by an N-acylhomoserine lactone autoinducer." Journal of Bacteriology. 177.17 5000-08. (1995)
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