Our Contribution
Our kill switch is based on a quorum sensing module found in Vibrio fischeri. It is the luxR-luxI system. In order to better design our experiments, we researched more about the luxI gene and its products. This information was added to the relevant part page, BBa C0061. We hope that this information can save future teams time and improve their experiments. We also have included additional articles in the references section that were supplemental in understanding and using a quorum sensing module.
Acyl-Homoserine-Lactone Synthase
*Reaction Catalyzed:
fatty acyl-[ACP] + S-adenosyl-L-methionine <=> an N-acyl-L-homoserine lactone + H(+) + holo-[ACP] + S-methyl-5'-thioadenosine
*Derived from Expasy by the Swiss Institute of Bioinformatics
In a 1996 study done by the University of Iowa and the University of Illinois, the optimal substrates for acyl homoserine lactone synthase, which is the direct product of the luxI gene, were shown to be S-adenosylmethionine (SAM) and hexanoyl-acyl carrier protein (hexanoyl-ACP). In experiments with many other similar substrates, the enzyme showed a high level of specificity for these substrates (Table 1) (Schaefer et al.).
The final conclusion of this paper indicated that this data on specificity provided direct evidence for the mechanism of the reaction catalyzed by acyl homoserine lactone synthase (AHL) to form acyl homoserine lactone signals. The AHL synthase is an autoinducer that catalyzes the formation of the amide bond between the SAM and the hexanoyl-ACP. Then it catalyzes the formation of the signals through this acyl-SAM intermediate (Schaefer et al.). Metacyc derived the Km for each substrate to be 130 for SAM and 9.6 for 3-oxohexanoyl-[acp]. The optimal temperature for the reaction was 20-30 ℃ and the optimal pH was 7.8 (“MetaCyc Acyl-Homoserine-Lactone Synthase”).
Other important parameters:
According to a bionumbers entry, referencing Kaufmann et al. and Schaefer et al., the synthesis and degradation rates of 3OC6-homo-serine lactone in the lux system are 3,300 nM/hr and 0.108/hr, respectively (Moran, “Synthesis and Degradation Rate Constant of 3O - Bacteria Vibrio Fischeri - BNID 112005”).
The transport rate of AHL is 0.05/sec, (Moran, “Transport Rate Constant of AHL (Acyl Homo-Ser - Bacteria Vibrio Fischeri - BNID 112003”)).
Half maximal induction of quorum sensing by 3OC6-homoserine lactone is 25-50 nM, (Moran, “Half Maximal Induction of Quorum Sensing by 3 - Bacteria Vibrio Fischeri - BNID 112007”).
In an article on detecting AHL production, we found useful information on the substrates for luxR from V. fischeri and effective lab procedures for detecting AHLs. The luxR from V.fischeri responds to AHLs with C6 or C8 carbon chains whether they have 3-oxo substitutions or not. From previous info in this writing, we know that a C6 carbon chain is optimal (Ravn et al.). From other research we found that the fatty-acyl substrate is from fatty-acid biosynthesis through acyl-[ACP] and not from fatty-acid degradation through acyl-CoA (“ENZYME Entry: EC 2.3.1.184”).
In order to understand quorum sensing and the types of AHLs produced from a strain, it is key to use several bacterial monitoring systems to evaluate the full range of AHLs produced by a strain or population. Biological monitoring systems are a cost-effective and fast way to evaluate AHL-production when compared to the more commonly used advanced methods like NMR. Other effective lab methods for AHL-profiling and determining the kinetics of AHL-production are thin-layer chromatography and agar well-diffusion assays for quantifying AHLs from bacterial supernatants (Ravn et al.).
Key Articles to Understanding Quorum Sensing and References for the Information Above:
Cimolato, Chiara, et al. “Exploring Alternative Quorum Sensing Model Structures and Quorum Quenching Strategies.” BioRxiv, Cold Spring Harbor Laboratory, 7 July 2023, https://doi.org/10.1101/2023.07.07.548074. Accessed 10 Oct. 2023.
Dong, Shi-Hui, et al. “Structure-Guided Biochemical Analysis of Quorum Signal Synthase Specificities.” ACS Chemical Biology, vol. 15, no. 6, 4 May 2020, pp. 1497–1504. ACS Publications, https://doi.org/10.1021/acschembio.0c00142. Accessed 14 July 2023.
Kaufmann, Gunnar F., et al. “Revisiting Quorum Sensing: Discovery of Additional Chemical and Biological Functions for 3-Oxo- N -Acyl homoserine Lactones.” Proceedings of the National Academy of Sciences, vol. 102, no. 2, 27 Dec. 2004, pp. 309–314. PNAS, https://doi.org/10.1073/pnas.0408639102. Accessed 17 June 2022.
Moran, Uri. “Synthesis and Degradation Rate Constant of 3O - Bacteria Vibrio Fischeri - BNID 112005.” Bionumbers.hms.harvard.edu, Bionumbers, bionumbers.hms.harvard.edu/bionumber.aspx?id=112005&ver=4&trm=luxI&org=. Accessed 10 Oct. 2023.
Moran, Uri . “Transport Rate Constant of AHL (Acyl Homo-Ser - Bacteria Vibrio Fischeri - BNID 112003.” Bionumbers.hms.harvard.edu, Bionumbers, bionumbers.hms.harvard.edu/bionumber.aspx?id=112003&ver=5&trm=acyl+homoserine+lactone+synthase&org=. Accessed 10 Oct. 2023.
Moran, Uri . “Half Maximal Induction of Quorum Sensing by 3 - Bacteria Vibrio Fischeri - BNID 112007.” Bionumbers.hms.harvard.edu, Bionumbers, bionumbers.hms.harvard.edu/bionumber.aspx?id=112007&ver=5&trm=acyl+homoserine+lactone+synthase&org=. Accessed 10 Oct. 2023.
“Synthesis and Degradation Rate Constant of C4 - Bacteria Pseudomonas Aeruginos - BNID 112008.” Bionumbers.hms.harvard.edu, Bionumbers, bionumbers.hms.harvard.edu/bionumber.aspx?id=112008&ver=2&trm=rhlI&org=. Accessed 10 Oct. 2023.
Pai, Anand, and Lingchong You. “Optimal Tuning of Bacterial Sensing Potential.” Molecular Systems Biology, vol. 5, no. 1, 7 July 2009, p. 286. National Library of Medicine, https://doi.org/10.1038/msb.2009.43. Accessed 10 Oct. 2023.
Parsek, M. R., et al. “Acyl Homoserine-Lactone Quorum-Sensing Signal Generation.” Proceedings of the National Academy of Sciences, vol. 96, no. 8, 13 Apr. 1999, pp. 4360–4365. PNAS, https://doi.org/10.1073/pnas.96.8.4360. Accessed 10 Oct. 2023.
Ravn, Lars, et al. “Methods for Detecting Acylated Homoserine Lactones Produced by Gram-Negative Bacteria and Their Application in Studies of AHL-Production Kinetics.” Journal of Microbiological Methods, vol. 44, no. 3, 2 Apr. 2001, pp. 239–251. ScienceDirect, https://doi.org/10.1016/s0167-7012(01)00217-2. Accessed 10 Oct. 2023.
Schaefer, A. L., et al. “Generation of Cell-To-Cell Signals in Quorum Sensing: Acyl Homoserine Lactone Synthase Activity of a Purified Vibrio Fischeri LuxI Protein.” Proceedings of the National Academy of Sciences, vol. 93, no. 18, 3 Sept. 1996, pp. 9505–9509, https://doi.org/10.1073/pnas.93.18.9505. Accessed 1 Dec. 2019.
“MetaCyc Acyl-Homoserine-Lactone Synthase.” Metacyc.org, metacyc.org/gene?orgid=META&id=G-11135#tab=RXNS. Accessed 9 Oct. 2023.
“ENZYME Entry: EC 2.3.1.184.” Expasy, Swiss Institute of Bioinformatics, enzyme.expasy.org/EC/2.3.1.184. Accessed 10 Oct. 2023.