Biomedical Engineering Reference
In-Depth Information
minimal media (Kalsner et al. 1995) supplemented with 0.6 M sucrose as an
osmoticum. Selected transformants for overexpression of the mannosidase
were grown in liquid yeast-fructose-threonine (YFT) (5 g yeast extract;
2 g fructose; 12 g threonine; 10 ml 100X salt solution; 1 ml 1000X trace
elements; and 0.6 g NaNO
3
, per litre). Strains were maintained on CYM
agar and spore suspensions were obtained by washing cultured CYM
agar plates with 8 ml 0.001% Tween 80. Mycelium for DNA isolations was
obtained by inoculating 500 ml liquid CYM with 108 spores and incubating
24 hours at 30°C with constant agitation (200 rpm). Expression cultures
were grown by inoculating 50 ml liquid YFT media with 10
8
spores and
incubating at 30°C with constant agitation (200 rpm) for up to 72h.
Construction of Expression Vector
The α-1,2-mannosidase IB expression vector was created by
replacing the N-terminal type-II transmembrane region of the
A.
nidulans
α-1,2-mannosidase IB gene with a synthetic signal sequence
(MDRFLAVISAFFATAFAK) by tailed PCR amplifi cation and fusion to
the inducible
alc
A promoter. The synthetic signal coding sequence was
fused to the coding region corresponding to nucleotide +73 to +1619
of the
A. nidulans
α-1,2-mannosidase IB gene. This was then fused to
the 2002bp
A. nidulans alcA
promoter, in pUC18 vector (ANIBSEC).
The α-1,2-mannosidase IC expression vector was created by replacing
the N-terminal type-II transmembrane region of the
A. nidulans
α-1,2-
mannosidase IC (
mns
IC) gene (AF233287) with the synthetic signal
sequence, MDRFLGRHLGLLRHCLRQ by tailed PCR amplifi cation and
fusion to the inducible
alcA
promoter. The synthetic signal coding sequence
was fused to the coding region corresponding to nucleotide +91 to +1759
of the
A. nidulans
α-1,2-mannosidase IC gene. This was then fused to the
2002bp
A. nidulans alcA
promoter, in pUC18 vector (ANICSEC2).
Protoplasting and Transformation
Protoplasts were prepared according to the method of (Fincham 1989),
using Sigma Lysing Enzyme for cell wall digestion. Protoplasts of strain
T580 (uridine auxotroph) were co-transformed with 1 mg of the selectable
marker pJR15, which converts transformed cells to uridine prototrophy
and with 1 mg of ANIBSEC or ANICSEC2. Transformants were initially
screened for integration of pFB94 by selection on minimal media and
positive transformants were then transferred to individual complete media
plates. Conidia were collected using 8 ml 0.01% Tween-80 and conidia
were used for subsequent inoculations. Transformants were screened for
