Difference between revisions of "Part:BBa C0061"

Latest revision as of 00:27, 12 October 2023

autoinducer synthetase for AHL

Synthesizes 3OC₆HSL, which binds to LuxR.

The lux cassette of V. fischeri contains a left and a right promoter. The right promoter gives weak constitutive expression of downstream genes.This expression is up-regulated by the action of the Lux repressor, LuxR. Two molecules of LuxR protein form a complex with two molecules the signalling compound HSL. This complex binds to a palindromic site on the promoter, increasing the rate of transcription.

Usage and Biology

Enzyme for creating acyl-homoserine lactones from normal cell metabolites. All LuxI proteins direct the synthesis of specific acyl-HSLs and show sequence similarity.

Sequence and Features

The Alma IGEM 2023 kill switch was 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. We have added useful findings here. To find the source articles and useful resources for quorum sensing modules visit the contributions page on Alma IGEM's 2023 wiki.

• 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.).

• • Allergen characterization of BBa_C0061: Not a potential allergen

The Baltimore Biocrew 2017 team discovered that proteins generated through biobrick parts can be evaluated for allergenicity. This information is important to the people using these parts in the lab, as well as when considering using the protein for mass production, or using in the environment. The allergenicity test permits a comparison between the sequences of the biobrick parts and the identified allergen proteins enlisted in a data base.The higher the similarity between the biobricks and the proteins, the more likely the biobrick is allergenic cross-reactive. In the full-length alignments by FASTA, 30% or more amount of similarity signifies that the biobrick has a Precaution Status meaning there is a potential risk with using the part. A 50% or more amount of identity signifies that the biobrick has a Possible Allergen Status. In the sliding window of 80 amino acid segments, greater than 35% signifies similarity to allergens. The percentage of similarity implies the potential of harm biobricks’ potential negative impact to exposed populations. For more information on how to assess your own biobrick part please see the “Allergenicity Testing Protocol” in the following page http://2017.igem.org/Team:Baltimore_Bio-Crew/Experiments

For the biobrick Part:BBa_C0061, there was a 24.9 % of identity match and 50.3% similarity match to the top allergens in the allergen database. This means that the biobrick part is not of potential allergen status. In 80 amino acid alignments by FASTA window, no matches found that are greater than 35% for this biobrick. This also means that there is not of potential allergen status.

>Internal Priming Screening Characterization of BBa_C0061: Has 3 possible internal priming site between this BioBrick part and the VR primer.

The 2018 Hawaii iGEM team evaluated the 40 most frequently used BioBricks and ran them through an internal priming screening process that we developed using the BLAST program tool. Out of the 40 BioBricks we evaluated, 10 of them showed possible internal priming of either the VF2 or VR primers and sometime even both. The data set has a range of sequence lengths from as small as 12 bases to as large as 1,210 bases. We experienced the issue of possible internal priming during the sequence verification process of our own BBa_K2574001 BioBrick and in the cloning process to express the part as a fusion protein. BBa_K2574001 is a composite part containing a VLP forming Gag protein sequence attached to a frequently used RFP part (BBa_E1010). We conducted a PCR amplification of the Gag-RFP insert using the VF2 and VR primers on the ligation product (pSB1C3 ligated to the Gag + RFP). This amplicon would serve as template for another PCR where we would add the NcoI and BamHI restriction enzyme sites through new primers for ligation into pET14b and subsequent induced expression. Despite gel confirming a rather large, approximately 2.1 kb insert band, our sequencing results with the VR primer and BamHI RFP reverse primer gave mixed results. Both should have displayed the end of the RFP, but the VR primer revealed the end of the Gag. Analysis of the VR primer on the Gag-RFP sequence revealed several sites where the VR primer could have annealed with ~9 - 12 bp of complementarity. Internal priming of forward and reverse primers can be detrimental to an iGEM project because you can never be sure if the desired construct was correctly inserted into the BioBrick plasmid without a successful sequence verification.

For the BioBrick part BBa_C0061, the first location of the internal priming site is on the 155-149 base number of the BioBrick and on the 10-16 base number of the VR primer. The second location of the internal priming site is on the 339-333 base number of the BioBrick and on the 9-15 base number of the VR primer. The third location of the internal priming site is on the 98-104 base number of the BioBrick and on the 11-17 base number of the VR primer.

Functional Parameters: Austin_UTexas

BBa_C0061 parameters

Burden Imposed by this Part:

Burden is the percent reduction in the growth rate of E. coli cells transformed with a plasmid containing this BioBrick (± values are 95% confidence limits). This BioBrick did not exhibit a burden that was significantly greater than zero (i.e., it appears to have little to no impact on growth). Therefore, users can depend on this part to remain stable for many bacterial cell divisions and in large culture volumes. Refer to any one of the BBa_K3174002 - BBa_K3174007 pages for more information on the methods, an explanation of the sources of burden, and other conclusions from a large-scale measurement project conducted by the 2019 Austin_UTexas team.

This functional parameter was added by the 2020 Austin_UTexas team.