Escherichia coli dh5α

Single mutants of mCherry-Rac1 were generated using the site-directed mutagenesis. Mutations were introduced into the pmCherryC1-Rac1 vector in a PCR reaction using site-specific primers (Invitrogen) and high-fidelity Phusion DNA polymerase (NEB). Template DNA was digested by DpnI (NEB), and PCR product was transformed into competent DH5α Escherichia coli (NEB). Bacterial colonies were screened for presence of the desired mutation by DNA sequencing.

The genomic DNA of tested isolates was isolated according to the protocol described by He (2011) (link). The presence of the luxS gene was confirmed by PCR with primers specific to the inner region of the mentioned gene (primer set 1, Table 2). For the purpose of the sequence analysis, approximately 800 bp fragment containing the luxS gene was amplified with primer set 2 containing modified adaptors for restriction enzymes NcoI and EcoRI (Table 2). Subsequently, amplified fragment was cloned to the pGEM-T easy vector (Promega) via technique of the sticky ends. Subsequently, the ligation mixture was transformed into the competent cells of Escherichia coli DH5α (NEB, USA) by the routine heat-shock protocol described by Sambrook and Russell (2006) (link). Positive colonies of each sample were selected on Lysogeny agar (LB-A) (Hi-media, India) containing ampicillin (100 µg/ml), X-Gal (40 µg/ml) and IPTG (50 µg/ml) (all from Merck, USA).
The plasmid containing the fragment of interest of each sample was isolated by the GenElute™ HP Plasmid Miniprep Kit (Merck, USA), sequenced and the data were deposited to the NCBI database.

Molecular biology reagents and chemicals were purchased from Fisher Scientific, Sigma-Aldrich, GOLDBIO, or New England Biolabs, Inc., unless specified otherwise. Escherichia coli DH5α (New England Biolabs, MA) was cultured in Luria–Bertani broth. DNA sequencing was performed at ACGT (Wheeling, IL). Primers were ordered from Integrated DNA Technologies (Coralville, IA) and listed in Supplementary Data 2. Plasmid pSkunk3-BLA was purchased from Addgene (plasmid 61531). PacBio Barcoded Universal Primers (Part Number: 101-629-100) was purchased from Pacific Biosciences (Menlo Park, CA).

Escherichia coli DH5α, BL21, and C2566 were purchased from New England Biolabs (USA). E. coli DH5α was used for DNA preparation and cloning. The cells were grown in lysogeny broth (LB) (Difco Laboratories Inc.,USA) with appropriate antibiotic supplementation (25 ug/ml kanamycin and 34 ug/ml chloramphenicol) for strain maintenance and plasmid construction in the different E. coli strains. Three E. coli strains (DH5α, BL21, C2566) were used for part characterization, which was conducted at 37°C on LB agar plates with optimum time-course for colony formation. The sequences of the DNA parts used in this study are presented in Table S1. The promoter and RBS were selected from the Registry (http://parts.igem.org) and the terminators were from [4 (link)]. For the DNA part preparation, the duplex oligosynthesis method of Macrogen (Korea) was employed.