Biology Reference
In-Depth Information
molecule and to do so in a range of organisms. The chromophore is generated by
maturation of the side chains of amino acids 65-67 that are located within an
11-stranded
-barrel structure. By changing amino acids at positions 65-67, GFP
variants with increased brightness and shifted excitation and emission spectra have
been generated (
Cormack
et al.
, 1996; Scholz
et al.
, 2000
). Some of the resulting
palette of coloured variants, such as cyan (CFP) and yellow (YFP) fluorescent
proteins, can be distinguished spectrophotometrically, allowing simultaneous visu-
alization of several promoter activities in the same cell (
Stepanenko
et al.
, 2011
). The
folding kinetics and stability of GFP and its variants are features crucial to their use in
determining real-time promoter activity. Several fast-folding variants now exist in
which the chromophore is rapidly generated after GFP expression. This ensures that
measurement of fluorescence at high temporal resolution accurately reflects pro-
moter activity (
Cormack
et al.
, 1996
). High temporal resolution of promoter activity
also requires the ability to precisely measure the amount of reporter protein produced
in short-time intervals. This is achieved most accurately when the biological half-life
of the reporter protein is either significantly shorter (e.g. Lux) or longer (e.g. GFP)
than the duration of the time interval within which expression is measured. GFPmut3
is a fast-folding variant that is extraordinarily stable in
E. coli
and
B. subtilis
with estimated half-lives of more than 24 h and approximately 10 h, respectively
(
Andersen
et al.
, 1998
;
Botella
et al.
, 2011
). Therefore, it is particularly suited to
accurate measurement of promoter activity in bacterial systems where growth cycles
and promoter response times are considerably shorter than these half-life values.
Several additional features of GFP are worthy of consideration when choosing it
as a reporter for high-resolution kinetic analysis of promoter activity. (1) The
excitation wavelength required to generate GFP fluorescence often also generates
significant autofluorescence in biological material, resulting in a high background.
This can result in problems of low signal-to-noise ratios, especially when analyzing
less active promoters. (2) Many molecules of biological interest (e.g. antibiotics)
fluoresce at the wavelengths used to excite GFP, a feature that will complicate or
even prevent its use as a reporter protein when studying their effects on gene expres-
sion. (3) Formation of the GFP chromophore requires molecular oxygen, which
makes it of limited use in obligate anaerobic organisms. (4) GFP excitation can gen-
erate significant quantities of intracellular hydrogen peroxide, and cells expressing
this protein at high concentrations may be oxidatively stressed (
Tsien, 1998
).
b
3.3
Luciferase
Luciferase is a second reporter used for high-resolution global analysis of promoter
activity. Two types of luciferase protein are commonly used, firefly (
Photinus
pyralis
) and bacterial. Firefly luciferase uses luciferin as a substrate, oxidizing it to
oxyluciferin in a reaction that utilizes molecular oxygen and ATP, and liberates light
at 560 nm (
Wilson and Hastings, 1998; Fraga, 2008
). Bacterial luciferase is encoded
by the
luxCDABE
operon that is usually sourced from
Photorhabdus luminescens
,
Vib-
rio harveyi
or
Vibrio fischeri
. Bacterial luciferase catalyzes the oxidation of reduced
