presentación de powerpoint -...
TRANSCRIPT
![Page 1: Presentación de PowerPoint - 2014.igem.org2014.igem.org/files/presentation/Goettingen_Championship.pdf · Later on at House‘s Office. 1.5 Billion Fungal Infections in Humans/Year](https://reader036.vdocuments.net/reader036/viewer/2022071017/5fd04aca774f19056811e61d/html5/thumbnails/1.jpg)
“PepTag”
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It All Began with...
2
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It All Began with...
2
Looks like a Lung Infection…
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It All Began with...
2
Looks like a Lung Infection…
but Antibiotics don’t work!
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Later on at House‘s Office
3
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Later on at House‘s Office
1.5 Billion Fungal Infections in
Humans/Year
3
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Later on at House‘s Office
Candida causes around400,000 septicaemiawith a 40% mortality
rate
1.5 Billion Fungal Infections in
Humans/Year
3
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Later on at House‘s Office
A. fumigatus causes Aspergillosis
mortality rate as highas 90%
1.5 Billion Fungal Infections in
Humans/Year
Candida causes around400,000 septicaemiawith a 40% mortality
rate
3
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Later on at House‘s Office
1.5 Billion Fungal Infections in
Humans/Year
Candida causes around400,000 septicaemiawith a 40% mortality
rate
A. fumigatus causes Aspergillosis
mortality rate as highas 90%
3
![Page 10: Presentación de PowerPoint - 2014.igem.org2014.igem.org/files/presentation/Goettingen_Championship.pdf · Later on at House‘s Office. 1.5 Billion Fungal Infections in Humans/Year](https://reader036.vdocuments.net/reader036/viewer/2022071017/5fd04aca774f19056811e61d/html5/thumbnails/10.jpg)
“PepTag”
A small change for man, a giant pain for germkind!4
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Current TherapeuticStrategies
5
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Current TherapeuticStrategies
5
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Current TherapeuticStrategies
5
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Current TherapeuticStrategies
5
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Current TherapeuticStrategies
5
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Current TherapeuticStrategies
5
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Current TherapeuticStrategies
5
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Our Vision
6
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Our Vision
Make Fungi Visible to the Immune System
6
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Our Vision
Make Fungi Visible to the Immune System
6
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Our Vision
Make Fungi Visible to the Immune System
6
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Our Vision
Make Fungi Visible to the Immune System
6
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How to Make Fungal Cells Visible?
7
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How to Make Fungal Cells Visible?
7
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How to Make Fungal Cells Visible?
7
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How to Make Fungal Cells Visible?
7
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How to Make Fungal Cells Visible?
7
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How to Make Fungal Cells Visible?
7
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How to Make Fungal Cells Visible?
7
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How to Make Fungal Cells Visible?
PepTag
7
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How to Make Fungal Cells Visible?
PepTag
7
Immune System Modulator
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How to Make Fungal Cells Visible?
PepTag
7
Antibody + Signal Molecule
Immune System Modulator
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How to Make Fungal Cells Visible?
PepTag
7
Immune System Modulator Antibody + Signal Molecule
Radiolabel
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How to Make Fungal Cells Visible?
PepTag
7
Immune System Modulator Antibody + Signal Molecule Radiolabel
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¬ Located on outer surface
¬ No / little glycosylations
Criteria for Suitable Fungi Surface Proteins
8
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Candida albicans Saccharomycescerevisiae
Candida glabrata Toxoplasma gondii
Aspergillus fumigatus
Aspergillusnidulans
Selection of 35 Different Surface Proteins
9
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PCR Amplification
Cloning
attB1 GOI attB2
PCR and Cloning
10
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Yeast Two-Hybrid Assay
11
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Yeast Two-Hybrid Assay
1. Cultivation of Prey Library and Bait Yeast Strains
11
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Yeast Two-Hybrid Assay
1. Cultivation of Prey Library and Bait Yeast Strains
11
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Yeast Two-Hybrid Assay
1. Cultivation of Prey Library and Bait Yeast Strains
11
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Yeast Two-Hybrid Assay
1. Cultivation of Prey Library and Bait Yeast Strains
11
2. Mating of the twoYeast Strains
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Yeast Two-Hybrid Assay
1. Cultivation of Prey Library and Bait Yeast Strains
11
2. Mating of the twoYeast Strains
![Page 44: Presentación de PowerPoint - 2014.igem.org2014.igem.org/files/presentation/Goettingen_Championship.pdf · Later on at House‘s Office. 1.5 Billion Fungal Infections in Humans/Year](https://reader036.vdocuments.net/reader036/viewer/2022071017/5fd04aca774f19056811e61d/html5/thumbnails/44.jpg)
Yeast Two-Hybrid Assay
1. Cultivation of Prey Library and Bait Yeast Strains
11
2. Mating of the twoYeast Strains
![Page 45: Presentación de PowerPoint - 2014.igem.org2014.igem.org/files/presentation/Goettingen_Championship.pdf · Later on at House‘s Office. 1.5 Billion Fungal Infections in Humans/Year](https://reader036.vdocuments.net/reader036/viewer/2022071017/5fd04aca774f19056811e61d/html5/thumbnails/45.jpg)
Yeast Two-Hybrid Assay
1. Cultivation of Prey Library and Bait Yeast Strains
11
2. Mating of the twoYeast Strains
![Page 46: Presentación de PowerPoint - 2014.igem.org2014.igem.org/files/presentation/Goettingen_Championship.pdf · Later on at House‘s Office. 1.5 Billion Fungal Infections in Humans/Year](https://reader036.vdocuments.net/reader036/viewer/2022071017/5fd04aca774f19056811e61d/html5/thumbnails/46.jpg)
Yeast Two-Hybrid Assay
1. Cultivation of Prey Library and Bait Yeast Strains
11
2. Mating of the twoYeast Strains
![Page 47: Presentación de PowerPoint - 2014.igem.org2014.igem.org/files/presentation/Goettingen_Championship.pdf · Later on at House‘s Office. 1.5 Billion Fungal Infections in Humans/Year](https://reader036.vdocuments.net/reader036/viewer/2022071017/5fd04aca774f19056811e61d/html5/thumbnails/47.jpg)
Yeast Two-Hybrid Assay
1. Cultivation of Prey Library and Bait Yeast Strains
2. Mating of the twoYeast Strains
11
![Page 48: Presentación de PowerPoint - 2014.igem.org2014.igem.org/files/presentation/Goettingen_Championship.pdf · Later on at House‘s Office. 1.5 Billion Fungal Infections in Humans/Year](https://reader036.vdocuments.net/reader036/viewer/2022071017/5fd04aca774f19056811e61d/html5/thumbnails/48.jpg)
Yeast Two-Hybrid Assay
1. Cultivation of Prey Library and Bait Yeast Strains
2. Mating of the twoYeast Strains
11
![Page 49: Presentación de PowerPoint - 2014.igem.org2014.igem.org/files/presentation/Goettingen_Championship.pdf · Later on at House‘s Office. 1.5 Billion Fungal Infections in Humans/Year](https://reader036.vdocuments.net/reader036/viewer/2022071017/5fd04aca774f19056811e61d/html5/thumbnails/49.jpg)
Yeast Two-Hybrid Assay
1. Cultivation of Prey Library and Bait Yeast Strains
3. Mated CellsTransferred to Selection Medium
11
2. Mating of the twoYeast Strains
![Page 50: Presentación de PowerPoint - 2014.igem.org2014.igem.org/files/presentation/Goettingen_Championship.pdf · Later on at House‘s Office. 1.5 Billion Fungal Infections in Humans/Year](https://reader036.vdocuments.net/reader036/viewer/2022071017/5fd04aca774f19056811e61d/html5/thumbnails/50.jpg)
Yeast Two-Hybrid Assay
1. Cultivation of Prey Library and Bait Yeast Strains
3. Mated CellsTransferred to Selection Medium
11
2. Mating of the twoYeast Strains
![Page 51: Presentación de PowerPoint - 2014.igem.org2014.igem.org/files/presentation/Goettingen_Championship.pdf · Later on at House‘s Office. 1.5 Billion Fungal Infections in Humans/Year](https://reader036.vdocuments.net/reader036/viewer/2022071017/5fd04aca774f19056811e61d/html5/thumbnails/51.jpg)
Yeast Two-Hybrid Assay
1. Cultivation of Prey Library and Bait Yeast Strains
3. Mated CellsTransferred to Selection Medium
11
2. Mating of the twoYeast Strains
![Page 52: Presentación de PowerPoint - 2014.igem.org2014.igem.org/files/presentation/Goettingen_Championship.pdf · Later on at House‘s Office. 1.5 Billion Fungal Infections in Humans/Year](https://reader036.vdocuments.net/reader036/viewer/2022071017/5fd04aca774f19056811e61d/html5/thumbnails/52.jpg)
Yeast Two-Hybrid Assay
1. Cultivation of Prey Library and Bait Yeast Strains
3. Mated CellsTransferred to Selection Medium
11
2. Mating of the twoYeast Strains
![Page 53: Presentación de PowerPoint - 2014.igem.org2014.igem.org/files/presentation/Goettingen_Championship.pdf · Later on at House‘s Office. 1.5 Billion Fungal Infections in Humans/Year](https://reader036.vdocuments.net/reader036/viewer/2022071017/5fd04aca774f19056811e61d/html5/thumbnails/53.jpg)
In Our Lab
12
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In Our Lab
12
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Establishing Contact with Our Community
13
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Establishing Contact with Our Community
13
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Establishing Contact with Our Community
13
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Establishing Contact with Our Community
13
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Establishing Contact with Our Community
13
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Establishing Contact with Our Community
13
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Results are needed!
14
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15
Results 2nd Yeast-Two-Hybrid Assay
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Results 2nd Yeast-Two-Hybrid Assay
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Sequencing and Homology Modeling
16
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Sequencing and Homology Modeling
25 Amino AcidSequence
16
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Sequencing and Homology Modeling
25 Amino AcidSequence
Interacting PeptideGB1 Scaffold16
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Aspergillus fumigatus
PepTagαSun1
Label
In vivo Experiments
SignalMolecule
+Antibody
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Aspergillus fumigatus
PepTagαSun1
Label
In vivo Experiments
CalcofluorBright Field Labeled PepTag Overlay
SignalMolecule
+Antibody
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Are Specific PepTagsPossible?
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Are Specific PepTagsPossible?
18
Labeled PepTag
A. fumigatusαSun1 PepTag
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Are Specific PepTagsPossible?
18
Labeled PepTag
A. fumigatusαSun1 PepTag
A. nidulansαSun1 PepTag
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Yeast BioBricks
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Name Description Size (kb)
BBa_K1430007 Tool to tag yeast proteins with GFP 4304
BBa_K1430008 Tool to tag yeast proteins with mRFP 4685
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Yeast BioBricks
19
Name Description Size (kb)
BBa_K1430007 Tool to tag yeast proteins with GFP 4304
BBa_K1430008 Tool to tag yeast proteins with mRFP 4685
TEF2 ADH1
gene of interest
BamH1BglII
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Yeast BioBricks
19
Name Description Size (kb)
BBa_K1430007 Tool to tag yeast proteins with GFP 4304
BBa_K1430008 Tool to tag yeast proteins with mRFP 4685
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Use the PepTag as a Diagnostic Tool
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Ask the Experts
Dr. U. Groβ Dr. S. Krappmann
Dr. J. Wagener
University Medical Center Göttingen
University Medical Center Erlangen
Ludwig-MaximiliansUniversity of Munich
21
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Ask the Experts
Dr. U. Groβ Dr. S. Krappmann
Dr. J. Wagener
University Medical Center Göttingen
University Medical Center Erlangen
Ludwig-MaximiliansUniversity of Munich
21
High specificity
Reliability
Easy application
Low cost
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Ask the Experts
Dr. U. Groβ Dr. S. Krappmann
Dr. J. Wagener
University Medical Center Göttingen
University Medical Center Erlangen
Ludwig-MaximiliansUniversity of Munich
21
High specificity
Reliability
Easy application
Low cost
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Ask the Experts
Dr. U. Groβ Dr. S. Krappmann
Dr. J. Wagener
University Medical Center Göttingen
University Medical Center Erlangen
Ludwig-MaximiliansUniversity of Munich
21
High specificity
Reliability
Easy application
Low cost
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Ask the Experts
Dr. U. Groβ Dr. S. Krappmann
Dr. J. Wagener
University Medical Center Göttingen
University Medical Center Erlangen
Ludwig-MaximiliansUniversity of Munich
21
High specificity
Reliability
Easy application
Low cost
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Ask the Experts
Dr. U. Groβ Dr. S. Krappmann
Dr. J. Wagener
University Medical Center Göttingen
University Medical Center Erlangen
Ludwig-MaximiliansUniversity of Munich
21
High specificity
Reliability
Easy application
Low cost
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Outlook Make It Even Faster!
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Outlook Make It Even Faster!
22
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GFP ScaffoldsFragmented in 3
Regions
Using a GFP Scaffold
23
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GFP ScaffoldsFragmented in 3
Regions
Using a GFP Scaffold
23
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GFP ScaffoldsFragmented in 3
Regions
Using a GFP Scaffold
23
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Fusion PCR
GFP ScaffoldsFragmented in 3
Regions
Using a GFP Scaffold
23
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Fusion PCR
Plasmidconstruction
GFP ScaffoldsFragmented in 3
Regions
Using a GFP Scaffold
23
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Ultimate Goal
24
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Ultimate Goal
24
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Ultimate Goal
24
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Ultimate Goal
24
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Ultimate Goal
24
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Ultimate Goal
24
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Ultimate Goal
24
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What about Emma?
25
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What about Emma?
25
It’s Fumigatus!
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Special Thanks to...
26
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Questions?
27