Biotechnol. Prog., 1999, Vol. 15, No.
2 3A
BIOTECHNOLOGY NOTABLES
Direct Transformation of Hairy
Roots
The direct gene transfer of
the green fluorescent protein (GFP)
reporter gene into hairy roots
has been described. Agrobacterium
tumefaciens, containing the Cauli-
flower Mosaic Virus 35S promoter,
is used to generate Hyoscyamus
muticus hairy roots with the kana-
mycin resistance and GFP genes.
The roots were placed onto callus-
inducing media to promote cell divi-
sion and regeneration. Transformed
root cells were selected on medium
containing 50 and 100 mg/L kana-
mycin and screened by visual in-
spection for GFP expression. Highly
fluorescent GFP-transformed root
cell clones were incubated in phyto-
hormone-free medium for the regen-
eration of the hairy root phenotype.
PCR amplification was used to con-
firm the presence of the GFP gene.
The results demonstrated that GFP
is not toxic to H. muticus plant tis-
sue, and minimal autofluorescence
allowed clear observation of GFP
fluorescence. This direct transfor-
mation procedure can be applied for
genetic manipulation of existing
hairy root cultures which have been
previously characterized and
selected for desirable physiological
traits.
Curtis et al., Page 278
Capture of Rare Cells in
Suspension
A method has been demonstrated
for separating a subpopulation of
rare cells from a larger cell popula-
tion. Polystyrene beads (99-170
µm) coated with antibody to CD45
were used to capture cells from a
target population of murine B-cell
hybridomas. Beads and cell suspen-
sions were loaded and periodically
inverted based upon the average
bead settling velocity, 0.027 cm/s for
99 µm beads and 0.079 cm/s for 170
µm beads. Captured cells were
counted directly on the bead sur-
face. The number of cells captured
per bead appeared to peak within
1-2 h, with the efficiency of capture
decaying after the first hour. These
results suggested that a part of the
target subpopulation was more sus-
ceptible to capture and that the
bead surface was not uniform in its
cell-binding capacity. Capture of
cells was effective out to dilutions of
1:10 000, with purity in the cap-
tured population of better than
74%. A theory that estimates the
frequency of cell-surface collisions
has been developed.
Leonard et al., Page 238
Measurement of Bacterial
Flagellar Force
A negative dielectrophoretic force
gradient has been used to estimate
the force produced by bacterial fla-
gella. A spatially-dependent dielec-
trophoresis (DEP) force funnel has
been generated in which bacteria
move randomly until the forward
force generated by the flagellar
motor is exactly equal and opposite
to the DEP force. When this occurs
the bacteria become motionless.
Numerical simulations of the elec-
tric field gradient were used to esti-
mate the electric field gradient as a
function of radius from the electrode
center. To obtain the force, the size
of the bacterial collection pattern
was compared with a 2-D simula-
tion of the electric field. Using this
technique, the motor force of Salmo-
nella typhimurium was measured to
be approximately 0.37 pN. This
quick and noninvasive method can
be used in high-conductivity media
(such as growth media) where the
organism will experience negative
DEP over a wide range of frequen-
cies.
Hughes and Morgan, Page 245
In Situ Chemostat NMR
Performance tradeoffs and the
effect of different mixing regimes
have been analyzed to quantify the
impact of device design decisions on
in situ nuclear magnetic resonance
(NMR). A method is described for
growing cells in a chemostat and
shortening the time required to
acquire informative in situ NMR
spectra from cell cultures. Perfor-
mance tradeoffs that can occur
among net signal, spectral resolu-
tion, and oxygenation due to sam-
pling volume, conductivity, gas
bubble, and fluid flow effects have
been theoretically analyzed. The
results were found to concur with
data from cell-free NMR experi-
ments performed in 18 mS/cm con-
ductivity medium. A redesign of the
NMR bioreactor realized gains
which enabled the acquisition of
spectra from dilute (3-4% (v/v))
Saccharomyces cerevisiae cultures
in 6.9 min with high resolution in
both the orthophosphate and the
β-NTP regions. Such gains included
lower conductive losses, better mag-
netic field homogeneity, and the
exclusion of gas bubbles from the
sampling zone. Samples with 16%
(v/v) cells yielded useful spectra
within 0.5-1.0 min.
Domach et al., Page 185
Metabolic Pathways of
Propionibacterium
End product formation and meta-
bolic regulation in the aerobic culti-
vation of Propionibacterium have
been investigated. Oxygen-supply-
shift cultivation was conducted in
which anaerobic cultivation was
shifted to aerobic cultivation once
exponential growth was attained.
Organic acids were determined
before and after the shift. Enzyme
activities and organic acid forma-
tion were determined by HPLC and
enzyme analysis. By these methods,
citrate and citrate synthase could
not be detected, indicating that the
TCA cycle was not utilized by Propi-
onibacterium. Enzyme analysis
indicated that the regulation of
organic acid formation was depend-
ent upon the molecule serving as
electron acceptor in the respiratory
chain reactions. During aerobic cul-
tivation, the results indicated that
oxygen replaced fumarate as the
final electron acceptor. The fate of
13C-labeled propionate was traced
with 13C NMR spectroscopy. These
results demonstrated the formation
of pyruvate in accordance with the
decomposition of propionate, sug-
gesting that the randomizing path-
way occurring in anaerobic growth
worked in a reversed direction in
the presence of oxygen.
Shimizu et al., Page 201