MAS5 transformation algorithm used
One millilitre of culture was withdrawn and 1ml of solution containing the appropriate concentration of antibiotic (F:T 1.25mg/L, fosfomycin 1mg/L, tobramycin 0.25mg/L and Control - sterile water) was added in triplicate under both aerobic and anaerobic conditions. The cultures were then incubated with shaking for a further one hour.
CM6 was inoculated into Mueller Hinton Broth (MHB), incubated under overnight and adjusted to an OD550nm of 1.0. This culture was then diluted 1/50 into 24 x 100mls MHB + 1% potassium nitrate (KNO3), with 12 grown for 18 hours with shaking under aerobic and 12 with shaking under anaerobic conditions until they reached early exponential phase (OD550nm = 0.4).
(Parameters: start time = 18, time unit = hours, min temperature = 37, temperature unit = C, media = Mueller Hinton Broth (+1% KNO3))
Ten millilitres of culture was removed and centrifuged for 12 minutes at 3,220g at 4oC to harvest cells. The supernatant was removed and cell pellets were stored on ice before immediately proceeding with RNA extraction. RNA was isolated using Trizol® reagent (Invitrogen, UK). Briefly, cell pellets were resuspended in 1ml Trizol® and chloroform before being centrifuged to separate the phases. The upper (aqueous) phase was then precipitated using sodium acetate, glycogen, and molecular pure 100% ethanol. The resulting pellet was washed three times in cold 70% ethanol and resuspended in 81µl of diethyl pyrocarbonate (DEPC)-treated water before immediately proceeding with DNAase digestion. DNAase digestion was performed using the TURBO DNAfree™ DNase Treatment kit (Ambion, UK) according to the manufacturer’s instructions. Briefly, 8µl of 10x Turbo DNAse buffer and 2µl Turbo DNAse was added to the RNA solution. The samples were incubated for 37oC for 30 minutes before resuspended DNAse inactivation reagent (9µl) was added and mixed. Samples were then incubated for 5mins at room temperature and finally, samples were centrifuged at 10,000g for 1.5mins and the supernatant transferred to a new RNAase free centrifuge tube which was stored on ice before proceeding directly to RNA cleanup. RNA cleanup was performed with the Qiagen RNAeasy® kit (Qiagen, UK) using the manufacturer’s cleanup protocol. RNA was eluted with 40µl DEPC-water which was added to the spin column membrane, incubated at room temperature for 5 minutes and then centrifuged at 13,200g for 1 minute to elute the RNA. The eluate was then reapplied to the spin column membrane and incubated at room temperature for a further 2 minutes before centrifugation at 13,200g for a further 1 minute. RNA was stored on ice before being analysed spectrophotometrically using a FLUOstar Omega microplate reader (BMG Labtech, Germany), using the LVis® plate and 1µl of RNA. Absorbance was measured at 230nm, 260nm, 280nm and 340nm. Concentration of RNA was calculated on the MARS data analysis software with 1 absorbance unit at 260nm being equivalent to 40µg/ml RNA. Purity was also assessed using A260/230 and A260/280 ratios to check for protein, solvent and salt contamination. RNA was split into two 20µl aliquots and immediately stored at -80oC.
(Parameters: Extracted product = total_RNA, Amplification = none)