Genesys 140 visible spectrophotometer

For the generation of
Phd/Doc-expressing Salmonella strains, the previously
described S. Typhimurium
(14028) phd-doc::Km strain with a knockout of the
endogenous Phd–Doc system was used.^{10 (link)} A group of pCA24N plasmids was generated by inserting the sequences
of full-length wild-type Phd_STm antitoxin, Phd_STm antitoxin variants containing selected single alanine substitutions
or sequences equivalent to selected Phd_STm peptides with
expression starting at Met52. Each of these plasmids (including an
empty pCA24N vector) were cotransformed with a Doc_STm-expressing
pBAD33 plasmid into the aforementioned Salmonella strain, enabling coexpression of Doc_STm (inducible expression)
and different Phd_STm constructs (basal leaky expression).
The cotransformations above were also performed with an empty pBAD33
plasmid, as a control for the absence of Doc_STm expression.
For the growth rescue experiment, overnight (1% tryptone, 0.5%
yeast extract, 0.5% NaCl, 1% glucose, 100 μg/mL of carbenicillin,
and 34 μg/mL of chloramphenicol) cultures of the generated S. Typhimurium strains were diluted to an OD₆₀₀ of approximately 0.006 into fresh M9 minimal medium supplemented
with 0.5% or 1% arabinose, 0.4% glycerol, 0.4% casamino acids, 100
μg/mL of carbenicillin, and 34 μg/mL of chloramphenicol.
Cell growth at a given time was monitored at OD₆₀₀ with
a Genesys 140 Visible Spectrophotometer (Thermo Scientific).

Overnight 5-mL cultures of the WT and ypfP mutant were started from single colonies and grown at 37°C with shaking. Cells were pelleted, washed in PBS, and normalized to an OD₆₀₀ of 1.0. Cultures (100 mL) were inoculated to a starting OD₆₀₀ of 0.01 and grown anaerobically at 37°C in a Coy anaerobic chamber. Ten-milliliter volumes of the WT and ypfP cultures were collected at 0-, 2-, 4-, 6-, 8-, 12- and 24-h time points. A 1-mL volume of each time point sample was used to measure OD₆₀₀ with a Genesys 140 Visible spectrophotometer (Thermo Fisher Scientific). The remaining 9 mL of sample was pelleted, and the supernatant was filtered. The pH of the sterile supernatant was measured using a FiveEasy Plus FEP20 pH meter (Mettler Toledo).