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Scientific
Publications - Work Done by Microbiology Reader
Supplementary information Yeast phenomics on a genome-wide scale
Strains.
Deletion strains in the BY4741
background, wild-type genotype MATa;his3D1;leu2D0;met15D0;ura3,
mutant genotype MATa;his3D1;leu2D0;met15D0;ura3,
ORF::kanMX4 were
provided by the EUROSCARF
stock centre. Strains were stored in 15% (w/v) glycerol solution at –80°C.
Media
and growth conditions: Strains
were inoculated in 350ml
of SD medium (0.14% yeast nitrogen base (YNB; Difco) without amino acids, 0.5%
ammonium sulphate and 1% succinic acid; 2% (w/v) glucose;
20mg/l histidine, 20mg/l methionine, 20mg/l uracil, 20mg/l lysine and 100mg/l
leucine, pH 5.8) and incubated for ~72h
at 30C° on a micro-titre plate shaker (500rpm). This procedure was repeated
once (second inoculation~48h).
For experimental runs, strains were inoculated to an OD of 0.03 - 0.1 in 350ml of SD medium and cultivated for 48h in a Bioscreen analyzer C ( Calculation of growth variables: The
growth variables length of lag phase, rate of growth and stationary phase OD
increment were calculated in the following way: The average blanking value of
0.067 (representing the background absorption of SD medium) was subtracted from
each OD measurement. All OD values were calibrated according to the formula
calibrated OD (ODc) =OD + 0.8324*OD3. Growth curves were
smoothened by averaging over 1h (3 measurements) to prevent digital noise from
affecting calculations. The effects of catastrophic events were reduced by
removing negative slopes; if any calculated slope between consecutive OD values
the latter value was adjusted as to equal the higher. Furthermore, if end OD
values did not equal at least a doubling of the initial OD no growth was
considered to occur. No rate of growth or stationary phase OD increment was
calculated and adaptation time for that curve was set to 48h. None such curves
were found among the NaCl data. Length
of lag phase was calculated in
the following way: Derived OD values were log transformed. 1 was added to avoid
negative values. Initial OD was taken as the mean of the five OD values. Slopes
were calculated between every 8:th value along the curve. Intercepts were
calculated between every slope and the initial OD. The mean of the 2 highest
slopes was taken as the length of the lag phase. Rate
of growth was calculated in the
following way: Derived OD values were log transformed. Slopes were calculated
between every third consecutive OD value. No slopes were calculated from the 8
first measurement points or from ODc < 0.1 to avoid possible
contribution from digitalisation effects. Of the seven highest slopes, the top
two were discarded as possible artefacts and a mean was calculated from the
following five. Generation time was calculated as log 2 divided by the mean. Stationary
phase OD increment was
calculated in the following way: Initial OD was determined as for length of lag
phase. Last measurement value was taken as the End OD. The difference between
End OD and initial OD was taken as the stationary phase OD increment value. If
the standard deviation for the last six measurements was more than 2% of End OD
the culture was not considered to have reached stationary phase and no
stationary phase OD increment was calculated from that curve. Calculation
of Phenotypic Index (PI): In
each Bioscreen Analyzer C and in each run, 8 wild-types (wt) were grown in the
same condition as the deletion strains. All 8 wt were used in the calculations
of the average wt values below. For the growth variable rate of growth, a
logarithmic strain coefficient, LSC,
for each strain, in each environment was calculated as:
where
wtkj
is the doubling time of the k:th
measurement of the wildtype in environment j,
xkj is the
doubling time of strain i in
environment j and r
indicates the run. The
logarithmic phenotypic index for each strain in NaCl, LPINaCl,
for rate of growth was calculated as:
LSC and LPINaCl, for length of lag phase and LSC for the stationary phase OD increment was calculated correspondingly. LPINaCl, for stationary phase OD increment was calculated as
Strain coefficient, SC, and phenotypic index, PI, were calculated in a similar manner. No log transformations were performed. Significance
tests: We performed tests of
the null hypothesis that LPINaCl,
equals 0 separately for lag phase, rate of growth and stationary phase and for
each environment/strain combination. Statistical significance of the data was
calculated using a two-tailed, two sample Students T-test (a=0.05,
df =2). In order not to reject the null hypothesis only because of a too small
variance estimate, a threshold of three mean standard deviations (a=0.0025)
was also applied. The threshold was rounded upwards to 0.12 for rate of growth,
0.20 for stationary phase OD increment and 0.25 for adaptation time when
considering the salt data and to 0.05 for rate of growth, 0.2 for stationary
phase and 0.2 for adaptation time when considering the synthetic medium data.
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