Gas permeable microplate adhesive film

Growth studies of bacterial strains were conducted using microplate reader kinetic assays. Overnight cultures were inoculated into 10 ​mL of LB medium from single colonies, and grown at 30 ​°C. These cultures were then diluted 1:100 into 500 ​μL of LB medium with appropriate concentrations of arabinose and p-coumarate in 48-well plates (Falcon, 353072). Plates were sealed with a gas-permeable microplate adhesive film (VWR, USA), and optical density and fluorescence were monitored for 48 ​h in a Biotek Synergy 4 plate reader (BioTek, USA) at 30 ​°C with fast continuous shaking. Optical density was measured at 600 ​nm. The amount of time necessary for the culture to reach an OD₆₀₀ of 0.16 was defined as the lag time.

BarSeq-based experiments utilized the P. putida RB-TnSeq library, JBEI-1, which has been described previously (Thompson et al., 2019a ). An aliquot of JBEI-1 was thawed on ice, diluted into 25 ​mL of LB medium supplemented with kanamycin and grown to an OD₆₀₀ of 0.5 ​at 30 ​°C. Three 1 ​mL aliquots of the library were pelleted and stored at −80 ​°C to later serve as the t₀ of gene abundance. Libraries were then washed in MOPS minimal medium and diluted 1:50 in MOPS minimal medium with 10 ​mM p-coumarate, ferulate, benzoate, p-hydroxybenzoate, protocatechuate, vanillin, vanillate, phenylacetate, or D-glucose. Cells were grown in 600 ​μL of medium in 96-well deep well plates (VWR). Plates were sealed with a gas-permeable microplate adhesive film (VWR, USA), and then grown at 30 ​°C in an INFORS HT Multitron (Infors USA Inc.), with shaking at 700 ​rpm. Two 600-μL samples were combined, pelleted, and stored at −80 ​°C until analysis by BarSeq, which was performed as previously described (Rand et al., 2017 (link); Wetmore et al., 2015 (link)). All fitness data are publicly available at http://fit.genomics.lbl.gov.