T. reesei - TNAU Genomics
T. reesei - TNAU Genomics
T. reesei - TNAU Genomics
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Protein quality control in the<br />
industrially - exploited fungal<br />
cell factory Trichoderma <strong>reesei</strong><br />
Prof Helena Nevalainen, Dr Liisa Kautto,<br />
Dr Jasmine Grinyer and Dr Junior Te’o<br />
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Prof Helena Nevalainen, brief biography<br />
FUNGAL BIOTECHNOLOGIST<br />
Research interests include molecular biology and functional<br />
proteomics of biotechnologically important filamentous fungi, in<br />
particular the industrially-exploited Trichoderma <strong>reesei</strong>. T. <strong>reesei</strong> offers<br />
a powerful cell factory for efficient expression of valuable gene<br />
products for various industrial and pharmaceutical applications. Other<br />
research interests feature fungal proteomics, biological control and<br />
molecular prospecting of the environment for novel bioactivities.<br />
Brief employment history:<br />
2009- Member of the Australian Research Council College of Experts<br />
2008- Head of the Department of Chemistry and Biomolecular Sciences, Macquarie<br />
University, Sydney<br />
2005- Professor of Biotechnology (Personal Chair) Department of Chemistry and<br />
Biomolecular Sciences, MU<br />
_____________________________________________________________________<br />
1993- present Consultant to ROAL Ltd Finland<br />
1992- present Adjunct Professor (Docent) in Applied Microbial Genetics, University of<br />
Helsinki, Faculty of Science<br />
1989-1992 Head, Microbiological Department, Research Laboratories of ALKO Ltd<br />
Helsinki, Finland<br />
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Trichoderma <strong>reesei</strong> as a cell factory<br />
• One of the best known cellulolytic fungi, producing<br />
extracellular proteins up to 100 g/L<br />
• Most of the secreted protein (~60%) is cellobiohydrolase I<br />
(CBHI)<br />
• Employed in industrial enzyme production<br />
• Developed for production of foreign gene products<br />
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southdakotapolitics.blogs.com/.../index.html<br />
http://www.sustainpack.com/images/aap/pulp_<br />
bleaching.jpg<br />
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T. <strong>reesei</strong> has a eukaryotic<br />
protein-processing<br />
machinery<br />
Problem with foreign proteins:<br />
yields 100-1000 x lower than<br />
those of endogenous proteins<br />
Misfolded proteins are recognised<br />
by the cellular QC mechanisms<br />
(UPR and ERAD) and degraded<br />
by the proteasome<br />
UPR<br />
ERAD<br />
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Our targets:<br />
The structure of the T. <strong>reesei</strong> proteasome (proteomics<br />
approach)<br />
Effect of overexpression of a dominant misfolded<br />
protein on gene expression (microarrays)<br />
Physiological effects of overexpression of a dominant<br />
misfolded protein (fluorescence and EM microscopy)<br />
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The 26S proteasome, a proteolytic<br />
macromolecule<br />
Chymotrypsin-like (ChTL) – 5<br />
Trypsin-like (TL) – 2<br />
Peptidylglutamylpeptide hydrolyzing (PGPH) – 1<br />
Purified by 2-step chromatography:<br />
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POROS Mono HQ - anion<br />
exchange<br />
Sephacryl S-500 HR - size<br />
exclusion<br />
Kautto et al. (2009) Prot. Expr. Purif. 67:156-163.<br />
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2-DE analysis of the purified 26S proteasome<br />
q first dimension by pI<br />
q second dimension by mass<br />
q spots analysed by tandem mass spectrometry<br />
protein mass<br />
3 pI 10<br />
Protein spot from<br />
2-D gel<br />
Tryptic digestion &<br />
peptide extraction<br />
TYGGAAR EHICLLGR<br />
GPGFK<br />
GANR PSTTGVEMFR<br />
Unmodified and<br />
modified peptides<br />
Protein identification<br />
by database matching<br />
624.3<br />
769.8<br />
893.4<br />
994.5<br />
1056.1<br />
1326.7<br />
1501.9<br />
1759.8<br />
1923.4<br />
2100.6<br />
600 2200<br />
MALDI mass<br />
spectrometry<br />
or MS/MS<br />
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2D-map of the T. <strong>reesei</strong> 26 S proteasome<br />
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Summary of protein identifications from the<br />
26S proteasome<br />
Total number of protein spots analysed 176<br />
Identifications by CSI 51<br />
Identifications from the in-house T. <strong>reesei</strong><br />
database<br />
45<br />
20S particle subunits 14<br />
19S particle subunits 4<br />
PIPs (proteasome interacting proteins) 9<br />
Chaperones 7<br />
No association to the proteasome 13<br />
A<br />
B<br />
.<br />
C<br />
20<br />
S<br />
19S<br />
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Making a misfolded<br />
protein (CBHI) by<br />
replacing cysteine<br />
residues with<br />
proline<br />
Disulfide bond First cysteine Location Second cysteine Location<br />
1 Cys4 L (Pca1-His11) Cys72 S (Asn70-Asp74)<br />
2 Cys19 S (Pro12-Ser20) Cys25 S (Thr24-Asp35)<br />
3 Cys50 S (Thr48-Asp52) Cys71 S (Asn70-Asp74)<br />
4 Cys61 L (Ser58-Asp63) Cys67 H (Asn64-Lys69)<br />
5 Cys138 L (Val133-Leu140) Cys397 L (Gly395-Val403)<br />
6 Cys172 L (Met149-Leu180) Cys210 Cys210<br />
7 Cys176 L (Met149-Leu180) Cys209 S (Gly205-Cys209)<br />
8 Cys230 S (Ser222-Thr231) Cys256 S (Thr255-Asp257)<br />
9 Cys 238 S (Glu236-Gly240) Cys243 L (Asp241-Gly254)<br />
10 Cys261 S (Cys261-Trp263) Cys331 H (Asp328-Phe338)<br />
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The expression vector<br />
cbh1 promoter (red)<br />
signal sequence (black)<br />
DNA encoding CBHI core region (blue)<br />
Venus gene (yellow)<br />
cbh1 transcription termination region (ttm: truncated terminator; ftm: full<br />
terminator; grey)<br />
hygromycin selection marker gene under the pki promoter (green)<br />
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PCR amplification of a mutant cbh1 gene (p4)<br />
Mutant genes transformed into T. <strong>reesei</strong> conidia by biolistic<br />
bombardment > strains with one copy of the gene in the cbh1<br />
locus<br />
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Expression of a misfolded CBHI<br />
causes physiological stress<br />
Mutations also affected the profiles of secreted proteins<br />
and cellulase activity<br />
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Genome-wide effects of the expression of a<br />
mutant CBHI<br />
Custom ArrayTM 12K slides<br />
Genome wide gene-specific<br />
microarray: 30-45 mer oligonucleotide<br />
probes representing<br />
the 9129 gene open reading<br />
frames (ORFs) derived from the<br />
Trichoderma <strong>reesei</strong> sequencing<br />
project (http://genome.jgipsf.org/Trire2/<br />
Trire2.home.html)<br />
CVt: Cy3-ULS;<br />
others: Cy5-ULS<br />
Set of UPR/ERAD related<br />
genes<br />
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jgi Trire Gene name U/E jgi Trire Gene name U/E jgi Trire Gene name U/E<br />
120153 19S RPN1 E 120650 20S ALPHA6 E 122396 NPL4 E<br />
78423 19S RPN2 E 76010 20S ALPHA7 E 131033 PEX4 E<br />
77591 19S RPN3 E 78882 20S BETA1 E 50647 HRD1 U<br />
82512 19S RPN4 E 53446 20S BETA2 E 64023 HRD3 U<br />
68304 19S RPN5 E 58125 20S BETA3 E 121977 PPI U<br />
54454 19S RPN6 E 78925 20S BETA4 E 52050 CPR3(CYPA) U<br />
49923 19S RPN7 E 121009 20S BETA5 E 122920 BIP U<br />
80843 19S RPN8 E 66707 20S BETA6 E 73678 CAL U<br />
77330 19S RPN9 E 105189 20S BETA7 E 119903 DER1 U<br />
66591 19S RPN10 E 22994 CDC48 E 35465 LHS1 U<br />
12189 19S RPN11 E 121397 SEC61 E 122415 PDI U<br />
48366 19S RPN12 E 80400 UCH1 E 28928 PRPA U<br />
73574 19S RPT1 E 21246 UFD1 E 119890 TIGA U<br />
77587 19S RPT3 E 72606 UBA1 E 64285 EDEM U<br />
63751 19S RPT4 E 123753 RUB1 E 119664 DOA4 U<br />
23206 19S RPT5 E 47635 SKN7 E 46902 HAC U<br />
78817 19S RPT6 E 123493 SSM4(DOA4) E 45242 IRE U<br />
121343 20S ALPHA1 E 123773 UBC1 E 81164 PTC U<br />
79825 20S ALPHA2 E 123559 UBC12 E 55362 HSP70 U<br />
73564 20S ALPHA3 E 77732 UBC6 E 119731 HSP60 U<br />
124031 20S ALPHA4 E 59987 UBC7 E 44504 ACT1 HK<br />
55644 20S ALPHA5 E 55788 UBC7homolog E 119735 GAPDH HK<br />
E: ERAD U: UPR HK: House keeping<br />
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An overview of gene expression<br />
Up- ( 1.5 fold) or down-regulated ( 1.5 fold) genes from<br />
the T. <strong>reesei</strong> at three time points: A. 12 h; B. 24 h and C.<br />
48 h<br />
Most of the differentially expressed genes belonged to<br />
the class ‘metabolic pathways’<br />
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Up- and down-regulated genes assigned to their functional<br />
classes in strain CVt4. Up-regulated ; down-regulated .<br />
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Expression changes in UPR/ERAD related genes<br />
RutC30 CVt2 CVt4 CVt5 RutC30 CVt2 CVt4 CVt5 RutC30 CVt2 CVt4 CVt5<br />
TR2<br />
code<br />
Gene Function 12h 12h 12h 12h 24h 24h 24h 24h 48h 48h 48h 48h<br />
122920 BIP UPR (3.669,<br />
0.543)<br />
(3.565,<br />
0.509)<br />
(4.4,<br />
0.505)<br />
(3.968,<br />
0.46)<br />
(3.667,<br />
0.644)<br />
(4.54,<br />
0.621)<br />
(4.257,<br />
0.664)<br />
(0.596,<br />
4.462)<br />
(0.652,<br />
2.931)<br />
(0.613,<br />
2.302)<br />
(0.574,<br />
2.814)<br />
(0.451,<br />
12.325)<br />
(0.368,<br />
7.251)<br />
(0.386,<br />
9.595)<br />
(0.407,<br />
8.18)<br />
122415 PDI UPR (5.237,<br />
0.395)<br />
(0.427,<br />
26.836)<br />
73678 CAL UPR (2.022,<br />
0.619)<br />
(1.603,<br />
0.631)<br />
(2.066,<br />
(0.612,<br />
1.998)<br />
(0.65,<br />
7.332)<br />
(0.541,<br />
3.923)<br />
0.669)<br />
(1.704,<br />
0.644)<br />
82512 19S RPN4 ERAD (11.622,<br />
0.445)<br />
(6.388,<br />
0.359 )<br />
68304 19S RPN5 ERAD (0.655,<br />
2.512)<br />
(0.667,<br />
2.081)<br />
80843 19S RPN8 ERAD (0.606,<br />
2.393)<br />
48366 19S RPN12 ERAD (0.569,<br />
1.876)<br />
76010 20S ALFA7 ERAD (0.602,<br />
2.412)<br />
(0.619,<br />
2.232)<br />
66707 20S BETA6 ERAD (0.578,<br />
1.539)<br />
121397 SEC61 ERAD (3.315,<br />
0.506)<br />
(0.612,<br />
5.107)<br />
(3.52,<br />
0.571)<br />
(3.792,<br />
0.533)<br />
(0.664,<br />
1.904)<br />
(0.628,<br />
4.888)<br />
(0.651,<br />
4.992)<br />
72606 UBA1 ERAD (0.577,<br />
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2.689)<br />
(1.816,<br />
0.665)<br />
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Summary<br />
Amongst the three CBHI mutant strains examined,<br />
CVt2 did not show major expression changes in the<br />
genes involved in the UPR and ERAD pathways<br />
CVt4 was showing a stress response at 48 h and<br />
CVt5 was already showing signs of stress at 12 h<br />
Only 2.5 to 6.3 % of the differentially expressed genes<br />
were common in each strain either at two time points or<br />
all three time points<br />
All mutant strains exhibited stress related physiological<br />
changes such as low biomass synthesis and thinner<br />
hyphae during cultivation when compared to the nonmutant<br />
CVt<br />
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Visualisation of the Trichoderma <strong>reesei</strong> proteasome<br />
A<br />
B<br />
C<br />
T. <strong>reesei</strong> hyphae immunolabelled with a polyclonal anti-yeast 20S antibody<br />
detected by Alexa Fluor®488 (green). ToPro3 used for nuclear staining (blue).<br />
A. 2 d old hyphae showing the 20S proteasome inside the hyphae<br />
B. Proteasomes in the conidia of 19 h old hyphae<br />
C. Proteasomes at the actively growing hyphal tip<br />
20<br />
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Co-localisation of CBHI to the proteasome<br />
A. B<br />
.<br />
C<br />
.<br />
White-resin embedded hyphae of T. <strong>reesei</strong><br />
labelled with monoclonal anti-CBHI (green) and<br />
polyclonal 20S antiserum (red)<br />
T. <strong>reesei</strong> Rut-C30: A. CBHI<br />
B. 20S proteasome<br />
C. Merged images<br />
A<br />
.<br />
B<br />
C<br />
T. <strong>reesei</strong> CVt: A. CBHI<br />
B. 20S proteasome<br />
C. Co-localisation (yellow)<br />
A<br />
B<br />
.<br />
C<br />
.<br />
T. <strong>reesei</strong> CVt4: A. CBHI<br />
B. 20S proteasome<br />
C. Co-localisation<br />
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Quantification of gold particles in thin sections with TEM<br />
5 nm (proteasome); 10 nm (CBHI)<br />
a, p
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Summary<br />
Co-localisation of mutant CBHI and 20S proteasome<br />
particles was most obvious in strain CVt4, where they<br />
seemed to aggregate on the ER membrane (CLSM)<br />
Distribution of the gold particles in the indirect<br />
immunolabelling of CBHI and the 20S proteasome was<br />
different in the mutant strain CVt4 compared to the nonmutant<br />
CVt and the non-transformant Rut-C30 (TEM)<br />
These findings would indirectly point to degradation of the<br />
mutant CBHI by the proteasome<br />
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Conclusions<br />
The T. <strong>reesei</strong> proteasome seems to have a very similar<br />
structure to proteasomes isolated from other eukaryotic<br />
cells<br />
The use of mutant (misfolded) forms of a native<br />
highly secreted protein to study cellular protein<br />
quality control is a new approach<br />
The heterologous protein Venus did not cause any<br />
stress to the strain in the culture conditions used when<br />
compared to the non-transformant Rut-C30<br />
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Conclusions cont…<br />
Of all the differentially expressed genes identified, 23-50%<br />
had no match to known genes or proteins, or hypothetical<br />
genes or proteins<br />
Expression of a mutant CBHI decreased transcription<br />
and CBHI activity<br />
The known ER-stress-induced chaperones, BiP, PDI<br />
and PPI were found to be affected in strain CVt4 (48 h)<br />
CVt5 responded by mainly up-regulating the genes<br />
involved in the ERAD pathway<br />
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Acknowledgements<br />
Australian Proteome Analysis<br />
Facility<br />
Liisa Kautto<br />
Jasmine Grinyer<br />
Junior Te’o<br />
Peter Bergquist<br />
Debra Birch<br />
MichaGodlewski<br />
Helsingin Sanomat<br />
Foundation<br />
Seppo Säynäjäkangas<br />
Foundation<br />
Australian Research<br />
Council<br />
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Macquarie University in a nutshell<br />
• Established in 1969<br />
• Located about 18 km Northwest from Sydney<br />
CBD<br />
• Good public transport (train, buses)<br />
• About 33,000 students, 30% international<br />
• 24 h campus security<br />
http://www.mq.edu.au/<br />
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Faculty of Science<br />
• 500 Staff (300 Academic, 100 Admin and 100<br />
Technical)<br />
• 500 HDR students<br />
• 3,500 Coursework students<br />
• Occupy 12 major buildings on campus<br />
• 10 National research centres<br />
• 7 Macquarie University research centres<br />
• More than 230 Labs<br />
28<br />
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Department of Chemistry and Biomolecular<br />
Sciences (CBMS)<br />
20 academic staff and 60 higher degree research<br />
students<br />
• Teachers are active researchers and supervisors<br />
• State-of-the-art laboratories and facilities<br />
• Personalised instruction: students receive individual<br />
attention and advice<br />
29<br />
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Analytical chemistry: advanced chemical and physical technologies<br />
Bio-organic and medicinal chemistry: drug discovery and design<br />
Chemical biology: application of structural chemistry to biomedical or<br />
biological problems<br />
Structural genomics: analyses of the 3-D structure and dynamics of<br />
proteins<br />
Proteomics: study of the thousands of types of proteins found in all life<br />
forms<br />
Functional glycomics: role of sugars in disease and infections<br />
Agricultural and plant cell biochemistry and biotechnology: metabolic<br />
processes in crop plants to help select new plant varieties<br />
Microbial genomics: vaccine and drug discovery, diagnostics,<br />
bioremediation<br />
Biotechnology: developing micro-organisms for production of<br />
commercially relevant proteins for biopharma and industry<br />
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Highly specialised instrumentation<br />
including:<br />
Fermentors for making gene products<br />
Flow cytometers for fluorescence based cell<br />
sorting<br />
NMR (Nucleic Magnetic Resonance) instruments<br />
for the studies of chemical and protein structures<br />
A range of mass spectrometers for protein and<br />
biomolecular identification and characterisation<br />
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Fermentors for making bioproducts<br />
www.oardc.ohio-state.edu/bioenergy<br />
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FACSAria cell sorting<br />
flow cytometer<br />
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A 600 MHerz<br />
NMR machine<br />
http://www.pharmacy.arizona.edu/faculty/yanglab/images/NMRFacility/NMR.JPG)<br />
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CBMS Centers<br />
• Australian Proteome Analysis Facility<br />
• Biomolecular Frontiers Research Centre<br />
• Macquarie University Centre for Analytical<br />
Biotechnology<br />
• Environmental Biotechnology Co-operative Research<br />
Centre<br />
• Grain Foods CRC<br />
• Macquarie NMR Facility<br />
• Macquarie University VisLab<br />
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Biomolecular Frontiers Research Centre<br />
• Mission: conduct world-class research into proteomics, glycomics<br />
and genomics and to enable a holistic understanding of organisms<br />
(systems biology)<br />
• Specific areas of research:<br />
• Cell biology (human, animal, plant and microbe)<br />
• Cellular interactions<br />
• Human disease biomarker discovery<br />
• Agri-food quality trait discovery<br />
• Protein post-translational modification and expression<br />
• Microbial physiology and pathogenicity<br />
• Lateral gene transfer and evolution<br />
• Bioinformatics<br />
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Proteomics to identify what membrane<br />
proteins socialise in cancer metastasis<br />
(identify binding partners of uPAR)<br />
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1. Dysregulation of proteolytic machinery<br />
at invasive edge of tumours (uPAR)<br />
2. Changes in cell adhesive properties as<br />
cancer becomes malignant (integrins)<br />
3. Differential responses to existent<br />
growth signalling pathways changes in<br />
malignancy (TGFb etc)<br />
uPAR<br />
37<br />
Saldanha et al., J Proteome Res. 2007 6: 1016-1028.
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“Glycobiology is one of the 10 emerging technologies<br />
that will change the world." MIT 2003<br />
Glycomics @ MQ<br />
Cancer glycomics<br />
Profiling the oligosaccharide changes that<br />
occur on membrane proteins in cancer<br />
metastasis and drug resistance<br />
Innate immune system<br />
Comparison of the glycoproteins of human<br />
breast milk with cow’s milk – important<br />
antipathogenic factors<br />
Technology development<br />
Specific separation technologies for the<br />
analysis of recombinant glycoprotein drugs<br />
(e.g. erythropoetin) (in collaboration with SGE<br />
Analytical, Melborne and Bruker Daltonics,<br />
Bremen, Germany)<br />
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Mobile DNA and gene diversity<br />
Understanding the impact of horizontal gene transfer<br />
on bacterial evolution and antibiotic resistant<br />
pathogens<br />
Environmental genomics<br />
Bioprospecting for genetic and microbial diversity<br />
G<br />
H<br />
I<br />
IS<br />
DNA helicase II<br />
integrase<br />
dnaK<br />
orf<br />
33<br />
9<br />
orf<br />
18<br />
6<br />
cox<br />
transposase<br />
IS IS IS<br />
gyrB<br />
pol<br />
B<br />
J<br />
gyr<br />
B<br />
pol<br />
B<br />
dn<br />
aA<br />
94%<br />
integrase<br />
orf<br />
25<br />
3<br />
orf<br />
11<br />
4<br />
orf<br />
85<br />
orf<br />
16<br />
9<br />
orf<br />
15<br />
2<br />
xre<br />
lysR<br />
IS<br />
IS<br />
IS<br />
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High throughput genomic approaches to understanding microbial<br />
systems<br />
q Microarrays, phenotype arrays<br />
Microbial genomics<br />
q Genome sequencing, metagenomics<br />
q Bioinformatics<br />
Applications in vaccine and drug discovery, diagnostics,<br />
bioremediation, carbon sequestration and microbial evolution<br />
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Development of fungi as cell factories for the<br />
production of enzymes and biopharmaceuticals<br />
Where genomics meets proteomics<br />
Pulp bleaching, detergents,<br />
biofuels…<br />
Antibodies, hormones…<br />
Directed evolution of<br />
biocatalysts for<br />
enhanced performance<br />
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41
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Australian Proteome Analysis Facility<br />
http://www.proteome.org.au/<br />
Leading proteomics service provider, nationally and internationally,<br />
to both academia and industry<br />
Developer of new technologies for protein analysis, continually<br />
forming new collaborations with Australian and international<br />
biotechnology communities<br />
Aus $16 million investment from National Collaborative<br />
Infrastructure Strategy (NCRIS)<br />
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Selection of mass spectrometers<br />
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Selection of mass spectrometers<br />
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Masters programs available<br />
• Master of Biotechnology (12 months coursework)<br />
• Master of Biotechnology (12 months coursework and<br />
research)<br />
• Master of Biotechnology with Master of Commerce (18<br />
months)<br />
• Master of Laboratory Quality Management (12 months)<br />
• Master of Radiopharmaceutical Sciences (18 months,<br />
available in 2011)<br />
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45