Biology Reference
In-Depth Information
subtraction of blank values) by [0.18/(OD
977
OD
900
)]. This calculation can be
approximated by multiplying each OD
600
reading by 5 when a 100-
l culture is
grown in a 96-well plate. The excitation and emission settings for GFPmut3 are
485/20 and 528/20 nm, respectively. For YFP, the excitation and emission settings
are 500/27 and 540/25 nm, respectively, while for CFP, the settings are 420/50 and
485/20 nm, respectively.
B. subtilis
strains without GFP fusions have background autofluorescence, which
must be subtracted from the GFP emission values of promoter fusion-containing
strains before calculating promoter activities. The level of background autofluores-
cence is dependent on the size and shape of the cells and on the density of the culture.
To perform this correction, OD
600
and fluorescence data are collected for six cultures
of control strains (i.e. those without promoter fusions). A curve is plotted with
the OD
600
values on the abscissa and fluorescence values on the ordinate axes.
A polynomial trend line (order 4) is fit to the resulting curve and its equation is
determined. This equation is used to subtract background autofluorescence from
themeasured fluorescence of each fusion-containing strain at the same optical density.
Because strains with different genetic backgrounds can have different size, shape or
growth characteristics, and hence levels of autofluorescence, controls for each genetic
background should be grown on every plate. This polynomial-mediated subtraction
should also be performed on the five remaining control (non-fusion-containing)
strains to assess the efficacy of background subtraction. If necessary background sub-
traction can be further refined by subtracting the maximum autofluorescence values
that remain after the polynomial treatment of the control strains.
Some difficulties in accurately subtracting the background fluorescence can arise
if the optical density of the culture remains constant for an extended period of time or
if the optical density decreases. In these situations, as the same OD
600
value is being
obtained several times along the growth curve, more than one fluorescent value can
be assigned to the same optical density point.
Promoter activity is then calculated at each time point by dividing the derivative
of the background-corrected fluorescence by the OD
600
[(dGFP/dt)/OD
600
]. This
gives the amount of GFP synthesized in the designated time period normalized to
the cell density of the culture. As GFP is very stable in
B. subtilis
, this reading is
a real-time measurement of the activity of the promoter under study in each time
interval. Promoter activity curves can be smoothed by plotting the average of three
successive time points.
m
7.7
Visualization of promoter activity
The activity of hundreds of promoters can be established with relative ease for a vari-
ety of experimental conditions using the high-throughput procedures described
(
Botella
et al.
, 2010, 2011
). We have used heat maps to visualize and analyze expres-
sion trends in these large data sets. Heat Map Generator (HMG,
http://bioinf.gen.tcd.
heat map images. Extra functionality is included to facilitate the pre-processing of
