Documente Academic
Documente Profesional
Documente Cultură
ICL1 gene
constructing a double knock-out
Matthijs Dekkers
Supervisor: Els Mol
Mei/Juni 2005
Introduction
Candida albicans, a
Introduction
of many studies
Understanding
pathogenesis
Finding new targets
for antifungals
Dimorphism
Ability to switch
between yeast and
hyphal growth
Essential for its
pathogenicity
Allows escape from
macrophage
Phagocytosis
and neutrophils
Normally destroyed in the phagolysosome
C. albicans can use different carbon sources, by
rapidly adapting its metabolism
This allows C. albicans to grow hyphae and
perforate the plasma membrane of the
macrophage
Releases the fungal cell while killing the
macrophage
Glyoxylate Cycle
Glucose is the
preferred carbon
source in most
organisms
Glyoxylate cycle
allows use of C2
carbon compounds
Studies have shown
that genes are
upregulated after
phagocytosis
Isocitrate Lyase
ICL1
ARG4
ARG4
ARG4
ARG4
PCR
ARG4
recombination
ARG4
BWP-17
Verification of Transformants
PCR technique will be used to verify the
correct integration of the marker and thus
the knock-out
different combinations of marker specific
and ICL1 specific primers will give
definitive answer whether transformants
are ICL1 knock-outs
Verification of Transformants
FP
A4
ARG4
A5
RP
PCR
FP+A4
FP+RP
A5+RP
Results
Cassette amplification
Amplification of ARG4
and HIS1 Wendland
cassettes with ICL1
homologous primers
results in 1900bp and
1428bp fragments
Cassette amplification
Amplification of ARG4
and HIS1 Wendland
cassettes with ICL1
homologous primers
results in 1900bp and
1428bp fragments
PCR products will be
elongated and used in
transformation
HIS1
ARG4
1900bp
1400bp
First Transformation
2 transformations, 1
with HIS1, 1 with
ARG4 were done on
BWP17 and plated on
resp. SD -his and SD
-arg plates
resulting
transformants were
analyzed with PCR
First Transformation
FP
A4
ARG4
A5
RP
PCR
BWP17
First Transformation
FP
A4
ARG4
A5
RP
PCR
1536bp
BWP17
First Transformation
FP
A4
ARG4
A5
RP
PCR
BWP17
789bp
First Transformation
FP
A4
ARG4
A5
RP
PCR
ARG4:
2369
ICL1:
2152
BWP17
First Transformation
CMD1 and CMD2
were both verified
by PCR to be
icl1::ARG4 /ICL1
These will be used
for further
experiments in this
study
CMD1
icl1::ARG4 /ICL1
CMD2
Second Transformation
The second transformation proved to be
harder than the first
Histidine cassette integrated in genome,
but not in ICL1 locus
A new screening technique was used
A Different Approach
Transformants lacking
both ICL1 alleles should
show attenuated growth
on ethanol/acetate plates
All double transformants
were plated on:
YPD
YNB + 2% glucose
YNB + 2% acetate
YNB + 2% ethanol
A Double Knock-Out?
YPD
YNB + 2% acetate
Second Transformation
FP
A4
ARG4
A5
RP
PCR
1536bp
CMD1
CMD3
BWP17
Second Transformation
FP
A4
ARG4
A5
RP
PCR
CMD1
CMD3
BWP17
789bp
Second Transformation
FP
H5
HIS1
H4
RP
PCR
CMD1
CMD3
BWP17
415bp
Second Transformation
FP
H5
HIS1
H4
RP
PCR
400bp
CMD1
CMD3
BWP17
Second Transformation
FP
HIS1
RP
FP
ARG4
PCR
2369bp
CMD1
CMD3
RP
BWP17
1781bp
A Double Knock-Out
icl1::ARG4 /icl1::HIS1
CMD3
icl1::ARG4 /icl1::HIS1
Future Research
Complementation of CMD3 with ICL1
Growth comparison on different media
wild type
BWP17
CMD1, CMD2 and CMD3
complemented CMD3
Acknowledgements
thanks to everybody, especially
Els Mol