Isopropyl β d thiogalactopyranoside iptg

B. subtilis and E. coli strains were grown in lysogeny broth (LB) (10 g tryptone, 5 g yeast extract, 5 g NaCl per L) or on LB plates fortified with 1.5% Bacto agar at 37°C. When appropriate, antibiotics were included at the following concentrations: 10 μg/ml tetracycline, 100 μg/ml spectinomycin, 5 μg/ml chloramphenicol, 5 μg/ml kanamycin, and 1 μg/ml erythromycin plus 25 μg/ml lincomycin (mls). Isopropyl β-D-thiogalactopyranoside (IPTG, Sigma) was added to the medium at the indicated concentration when appropriate.
For quantitative swarm assays, strains were grown to mid-log phase (OD600 0.3–1.0) concentrated to an OD₆₀₀ of 10 in PBS pH 7.4 (0.8% NaCl, 0.02% KCl, 100 mM Na₂HPO₄, and 17.5 mM KH₂PO₄) plus 0.5% India ink. LB plates fortified with 0.7% agar were dried for 10 min open-faced in a laminar flow hood and subsequently inoculated by spotting 10 uL cell resuspensions onto the center of the plate. Plates were dried an additional 10 min open-faced in a laminar flow hood and then incubated at 37°C in a humid chamber. Swarm radius was measured along the same axis every 30 minutes.
Images of swarm plates were obtained by toothpick-inoculating a colony into the center of an LB plate fortified with 0.7% agar. Plates were dried open-faced in a laminar flow hood for 12 min and incubated at 37°C in a humid chamber for 20 hrs. Images were taking using a BioRad Gel Doc.

The process of DSA using electroporated oligos has been described^{14 (link)}. Briefly, liquid cultures of E. coli BL21-AI cells (Thermo) harbouring a plasmid expressing Cas1 and Cas2 under the control of a T7-lac promoter (pWUR 1 + 2, which was a generous gift from U. Qimron) were started from plates and grown overnight in LB. In the morning, cultures were diluted 1:30 in 3 ml fresh LB containing L-arabinose (Sigma-Aldrich) at a final concentration of 0.2% (w/w) and 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG; Sigma-Aldrich), unless otherwise noted, and grown for an additional 2 h. Cells were then pelleted, re-suspended and washed in water three times to remove residual media. Cells were then resuspended in 50 μl (per 1 ml of the 3 ml culture) of water containing the psAA33 forward and reverse oligo strands each at a concentration of 3.1 μM and electroporated with a Bio-Rad Gene Pulser set to 1.8 kV, 25 uF and 200 Ω. Immediately following electroporation, cells were re-suspended in fresh LB and allowed to recover overnight. In the morning, the cultures were pelleted and frozen at −20 °C until DNA extraction.