N 3 oxooctanoyl l homoserine lactone 3oc8 hsl

To evaluate the HSL lytic activity of the purified AiiM, an analytical assay was developed in an Alliance HPLC system (Waters, United States) with a Symmetry (Waters, United States) C18 Column (75 mm, 3.5 mm). Both short and long acylated chains were included, 1 mM N-butyryl-DL-homoserine lactone (C4-HSL; Sigma Aldrich 09945), 1 mM N-(3-oxooctanoyl)-L-homoserine lactone (3OC8-HSL;Sigma Aldrich O1764), 1 mM N-decanoyl-DL-homoserine lactone (C10-HSL; Sigma Aldrich 17248), 1 mM N-(3-oxodecanoyl)-L-homoserine lactone (3OC10-HSL; Sigma Aldrich O9014), and N-(3-oxododecanoyl) homoserine lactone (3OC12-HSL; Sigma Aldrich O9139). 60 mM NaOH was used as positive control since it can hydrolyze HSL molecules and the reaction was stopped with the addition of 2N HCl. Several concentrations, from 250, 100, 50, 25, 10, and 5 μg/mL of purified AiiM were tested. Time exposition was also varied; 24 h, 2 h, 1 h, 30 min, 20 min, 10 min, and 5 min. All experiments were performed in triplicate. Chromatographic conditions were: column temperature 25°C, sample temperature 25°C, injection volume 10 μL, flow rate 1 mL/min, detection 205 nm. Elution mixture was made with 50 mM phosphates buffer pH 2.9:acetonitrile. For C4-HSL the relation was 90:10, 3OC8-HSL 60:40, C10-HSL 60:40, 3OC10-HSL 60:40, and 3OC12-HSL 50:50.

Escherichia coli BL21 (DE3) was used to achieve a heterologous expression of the AhlX protein in the pET28a (+) vector. The Agrobacterium tumefaciens NT1 strain expressing AHL-regulated LacZ was used to evaluate AHL activity with the bioassay method described by Dong et al. [9 (link)]. Erwinia carotovora SCG1 was provided by Prof. Ziduo Liu (HuaZhong Agricultural University, Wuhan, China) and was used to test the quenching activity of AhlX@Ni₃(PO₄)₂ for potato soft rot. Burkholderia glumae CCTCC AB 2,016,346 was purchased from the China Center for Type Culture Collection (CCTCC) and was used to test the quenching activity of AhlX@Ni₃(PO₄)₂ for rice seedling rot. N-(3-oxooctanoyl)-l-homoserine lactone (3OC8-HSL) was purchased from Sigma Aldrich (Missouri, USA). Other chemicals, if not specified, were purchased from Sinopharm Group Co. Ltd. (Shanghai, China).

Experiments were carried out with E. coli TOP10 cells transformed with plasmid constructs (Supplementary Table 3). Plasmid constructs were assembled to comply with the BioBrick RCF [10] standard using a variety of molecular cloning techniques. Biological parts were acquired from the registry of Standard Biological parts or from chemical synthesis. DNA sequences of assembled AHL-receiver devices are available for download (10.5281/zenodo.1252305). AHL inducers utilised were purchased as follows: N-Butyryl-DL-homoserine lactone {C4 HSL} (09945 Sigma), 3-oxohexanoyl-l-homoserine lactone {3OC6 HSL} (K3007 Aldrich), N-(3-Oxooctanoyl)-l-homoserine lactone {3OC8 HSL} (O1764 Sigma), N-(3-Oxododecanoyl)-l-homoserine lactone {3OC12 HSL} (O9139 Sigma), N-(3-Hydroxytetradecanoyl)-DL-homoserine lactone {3OHC14 HSL} (51481 Sigma) and N-(p-Coumaroyl)-l-homoserine lactone {pC HSL} (07077 Sigma). AHLs stock solutions were made in 100% DMSO.