GAMIDI
MTKD-CT-2005-029774
Prof. A. P. Moran, coordinator
Marie Curie Host Fellowship (ToK)
Dr. Sergey Ustinov
Laboratory of Molecular Biochemistry,
Department of Microbiology,
National University of Ireland, Galway
Information about Researcher:
Professional Experience:
Graduated: Novosibirsk Medical Academy (1998)
Research Scientist in the Institute of Internal
Medicine, Siberian Branch of the Russian
Medicine Academy of Sciences, Novosibirsk,
Laboratory of Molecular Genetics Therapeutic
Diseases.
George Mason University: Research assistant in
the Center for Biomedical Genomics and
Informatics, Fairfax, VA, USA
Project: “Fetal hemoglobin expression in
adipose tissue of morbidly obese”
Auburn University: Research fellow in the
Department of Pathobiology, AL, USA
Project: “Phage microarray”
Information about Researcher:
Areas of Expertise:
MICROARRAY TECHNIQUE:
Analysis of Gene Expression using cDNA microarray
technology: DNA/Protein/phage microarray. Microarray design.
Data analysis.
Experience in operating different microarray spotting robots.
MOLECULAR BIOLOGY:
RNA/DNA/proteins extraction from various tissues and
microorganisms: blood, myocardium, adipocytes, spleen, liver,
bone, gram negative and positive bacteria's.
PCR/RT-PCR, Real-Time/Quantitative PCR , sequencing (automatic
and manual, result processing), DNA/protein electrophoresis.
Experience using molecular biological software: Oligo, Vector-NTI,
DNA-star, MegAlign, Chromas, and others
Information about the project
Iron and pH-regulated gene expression of
Helicobacter pylori
To extend our knowledge of the process of
regulation and adaptation in H. pylori with
respect to two conditions; iron limitation and low
pH, which are known to oscillate within the
gastric mucosa, we are using cDNA microarrays
to identify genes whose expression are
dependant on changes in iron concentration
and pH.
Information about the project
Design of the Experiments and Methods
Growth conditions:
H. pylori strains 26695, J99 and AG1 were grown at 37°C under microaerobic
conditions on Columbia agar plates containing 5% horse blood and Dent’s
antibiotic supplement. Harvested growth from this medium is act as an inoculum for
liquid cultures in Brucella Broth containing 10% horse blood serum at 37°C.
Acid exposure experiments:
Bacteria from a liquid culture were harvested by centrifugation and were
resuspended in liquid culture whose pH has been adjusted to pH 5.0 with
hydrochloric acid. Incubation at 37°C under microaerobic conditions will continued
for 1 h.
Iron exposure experiment:
Iron-restricted conditions were achieved by supplementing the brucella broth with
20 uM desferal (desferrioxamine-B; Sigma). Iron-sufficient conditions will be achieved
by the absence of desferal.
Samples were subsequently used for RNA isolation and microarray analysis.
Information about the project
Design of the Experiments and Methods
Liquid culture
Liquid culture
Isolate total RNA
Isolate total DNA
Reverse Transcribe to
label with Cy3
cDNA, label with Cy5
Mix, Hybridize and Scan
Information about the project
Real-time PCR confirmation:
Unique primers will be designed for 100to 300-bp regions of selected up- and
down-regulated genes seen in the array.
Primer design will be aided by the Vector
NTI software.
Information about the project
Work in progress
Adjusted growing conditions.
RNA extraction protocol:
TRIzol® Max. Bacterial RNA Isolation Kit (Invitrogen) protocol was modified.
Gel electrophoresis of the purified Helicobacter Pylori RNA
shows distinct 16S and 23S ribosomal bands.
DNA extraction protocol:
Wizard® Genomic DNA Purification Kit (Promega) was modified.
Strain-specific PCR and Sequencing protocol:
Platinum® PCR SuperMix protocol, primers were designed by author.
QIAquick PCR Purification Kit (QIAGEN)
Confirmation of the strain (26695 and J99)
by the sequencing and strain-specific PCR:
Chromas and MegAlign software
Information about the project
Work in progress
Microaaray protocol:
The Bacterial Microarray Group
at St George's, University of
London (BµG@S)
The Helicobacter pylori array
has been designed to target the
two sequenced strains. The base
strain was selected as H.pylori
26695 and to this were
consecutively added extra
genes from strains J99
Future Prospects
Campylobacter jejuni gene expression in response to
iron limitation, growth temperature variation and
low-oxygen environment.
•There is a fast growing interest to the Campylobacter
jejuni as a zoonotic pathogen.
•Responsible for the majority of cases of bacterial
enteritis world-wide.
•Transmission to humans occurs through the
consumption of contaminated food, especially poultry,
milk and water.
•Campylobacter enteritis is characterised by the
destruction of the mucosal surfaces of the jejunum,
ileum and colon.
•Symptoms: diarrhea (can sometimes be bloody) also
extra-intestinal symptoms: fever, headache and
myalgia.
Future Prospects
Campylobacter jejuni gene expression in response to
iron limitation, growth temperature variation and
low-oxygen environment.
•C.jejuni array is a PCR product array based on the
NCTC 11168 strain annotation as described in Parkhill et
al (2000) Nature 403: 685-688.
•In addition to 1654 primer pairs designed to the NCTC
11168 strain, a further 73 primer pairs were designed to
amplify selected genes from strains 81116, 81176, 11351,
11828, 2523/90, 43431, 43432, 43438, 43449, 43456, 460,
8F169, P19 and CjX.
The Bacterial Microarray Group at St George's, University
of London (BµG@S)
Interesting points of collaboration with others in group
New type of microarray construction.
Analyzing the dynamic bacterial glycome with a lectin
microarray approach (Nature Chemical Biology 2, 153-157 (2006) )
lectin microarray
consisted of a panel of 21
lectins
a) Bacteria bind to the lectin microarray through the
interaction of bacterial surface carbohydrates with the lectin.
(b) Fluorescent labeling of bacterial strains and hybridization
to the array give distinct binding patterns.