Culture conditions and bacterial characterization
This protocol is extracted from research article:
Escherichia coli as a platform for the study of phosphoinositide biology
Sci Adv, Mar 27, 2019; DOI: 10.1126/sciadv.aat4872

Bacteria were grown in LB media supplemented with the appropriate antibiotics at 37°C for both plates and liquid cultures except for overnight cultures that were grown at 30°C to prevent the culture from reaching stationary phase. When small volumes of culture were needed, 6 ml was cultured in a 14-ml plastic tube. For larger cultures, we used Erlenmeyer flasks filled to 40% or less of their capacity to ensure proper aeration. Liquid cultures were grown in an orbital shaker at 260 rpm.

E. coli BL21 DE3 cells used for experiments were transformed with the appropriate plasmids, and a single colony was picked to grow overnight to an optical density at 600 nm (OD600) between 0.8 and 3. The overnight culture OD600 was measured, and the appropriate amount of culture was diluted in fresh media with the appropriate concentration of inositol required for the experiment to reach an OD600 of 0.05. With the exception of the time course experiments, the bacteria were then grown for 3 hours or less after the dilution to guarantee that all measures were taken during the exponential growth phase, thus minimizing cell-to-cell variability.

The morphology of the cells carrying each of the constructs was inspected visually by spreading 1 μl of bacterial culture in a glass slide, which was then covered with a coverslip and imaged in a bright-field inverted microscope using a 60× water objective with a 1.5× objective in the light path and a 10× magnification eye/camera piece (900× total magnification). For the growth rate characterization of the cells carrying the different phosphoinositide production constructs, we used a 96-well plate format, loading 300 μl of the freshly diluted culture per well. The plate was incubated at 37°C, and OD600 was measured every 30 min. Four wells (replicates) were used for each measure, and a correction was performed for the increase in OD600 due to evaporation observed in the control well. This correction turned out to be unnecessary; the results do not vary noticeably if it is omitted. The slope of the linear approximation to the middle of the exponential phase of growth was measured in each replicate and averaged afterward among the four replicates for each experimental unit. Since the path length of the OD600 measure is not 1 cm as defined by convention, note that the value obtained does not correspond directly to those of OD600 measured routinely but should show a linear correspondence with such. Since, for our purposes, we only intend to show the lack of growth defects due to the constructs that we use, only the relative (not the absolute) levels are relevant.

For protein extractions, we pelleted the culture equivalent to 1 ml of culture at an OD600 of 1 and resuspended it in lysis buffer consisting of the BugBuster 10X Protein Extraction Reagent from Millipore, diluted to 1× final concentration, and supplemented with rLysozyme Solution and Benzonase Nuclease (purity, >90%; also from Millipore) according to the manufacturer’s protocol. Protein gels were performed using the NuPAGE polyacrylamide system and transferred using the iBlot Dry Blotting System, both from Invitrogen, following the manufacturer’s protocols. The membrane was then probed with the horseradish peroxidase–labeled 9E10 monoclonal antibody against the myc tag obtained from OriGene (then Acris Antibodies). Western blots were imaged using the C-DiGit Chemiluminescence Western Blot Scanner and the WesternSure Chemiluminescent Substrate from LI-COR according to the manufacturer’s protocol.

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