modification, selection and production of cyclic...
TRANSCRIPT
![Page 1: Modification, Selection and Production of Cyclic Peptides2011.igem.org/files/presentation/Potsdam_Bioware_Championship.pdf · PowerPoint Presentation Author: iGEM11 Little Smurfs](https://reader036.vdocument.in/reader036/viewer/2022071015/5fce0a50cfae34408a33b099/html5/thumbnails/1.jpg)
iGEM 2011 World Championship Jamboree at MIT in Boston
Sunday, 6th November 2011
Modification, Selection and Production of Cyclic Peptides
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Outline
Laboratory
Introduction
Outreach & Xtras
2
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3
Potential of Cyclic Peptides
• every year, millions of people are infected with deseases related to protease activity
• cyclic peptides • stable • unique potential • use as therapeutic agent
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4
Microviridin Family
• cyclization over side chain residues – no cysteins or any backbone structure
involved
• cyclic peptide with two ester and one amide bond
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The Project Microcystis aeruginosa
mdn cluster
Library Generation
Selection System
Optimized Protease Inhibitor
Heterologous Expression in E. coli
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Modularization of the Cluster
6
• 6.5 kbp cluster with 5 important elements: • mdnA pro-peptide sequence • 4 different enzymes for processing of the
pro peptide
mdnC mdnB mdnA mdnD
BBa_K627005
mdnE
BBa_K627004
mdnD
BBa_K627002
mdnB
BBa_K627001
mdnA
BBa_K627003
mdnC
mdnE
ligase ligase acetyltransferase ABC transporter
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Characterization
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0
100000
200000
300000
400000
500000
600000
700000
3 8 13 18
mA
U a
t 2
10
nm
t in min
1800
m/z
2100 1500 1200 900 2400
1734.12 Da
+22 Da
+38 Da
-368 Da -766 Da
Mass Spectrometry HPLC
mdnC mdnB mdnA mdnD mdnE
YGGTFKYPSDWEDY
successfull detection and characterization
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Design of mdnA Libraries
8
0
5
10
div
ers
ity
• Library successfully generated
wt aa Y G G T F K Y P S D W E D Y
modified tht gvt gvt ACC nkk AAA TAC CCT TCT GAC TGG GAA GAT tht
aa diversity
Y G G F Y
V A A A V F D D … F
W
Total Diversity: 810 possible mutants
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The Progress Microcystis aeruginosa
mdn cluster
Library Generation
Selection System
Optimized Protease Inhibitor
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coating
Phage Display
10
M13 Phage
Pili
mdnA-myc-geneIII
binding
Infected E.coli
washing
washing
elution infect E.coli
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11
Preparing the Phage
mdnA-myc-tag-geneIII
M13 Phage
gene III microviridin
myc-tag
mdnC mdnB mdnA-myc-geneIII mdnD mdnE
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Establishing Phage Display
12
BBa_K627006
mdnA-myc-gene III
BBa_K627007
myc-gene III
Generated BioBricks
ELISA
• myc tag detected
0
0,2
0,4
0,6
0,8
1
1,2
control mdnA
exti
nct
ion
at
49
2 n
m
9E10 (anti-myc) Ab
anti-geneVIII Ab-HRP
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Test Panning
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Phage Display – Panning on Trypsin
0
1
2
3
4
5
6
7
pre-panning post panning
md
nA-P
hage
in %
• microviridin on phage
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Panning with Different Proteases
0
20
40
60
80
100
120
clon
e nu
mb
er a
fter
pa
nnin
g
• microvirdin binds papain
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The Progress Microcystis aeruginosa
mdn cluster
Library Generation
Selection System
Optimized Protease Inhibitor
![Page 16: Modification, Selection and Production of Cyclic Peptides2011.igem.org/files/presentation/Potsdam_Bioware_Championship.pdf · PowerPoint Presentation Author: iGEM11 Little Smurfs](https://reader036.vdocument.in/reader036/viewer/2022071015/5fce0a50cfae34408a33b099/html5/thumbnails/16.jpg)
In Vivo Selection
16
ssTorA
• Basic Idea • β–lactamase is only active in
periplasm • signal sequence of TorA serves
as a export signal
ampicillin
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Results
Resistenztest/Überleben Proof of principle? BioBricks
17
• functional export of β-lactamase into the periplasm worked cells were able to survive up to 400 µg/ml ampicillin
0%
20%
40%
60%
80%
100%
120%
140%
Surv
ivin
g C
FU
0
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In Vivo Selection
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proteolytic cleavage • cells will die
ampicillin
protease
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BBa_K627017
14 3C
19
Protease Detector at Work ?
Generated BioBricks
BBa_K627012
TorA-14 3C cleavage-β-lactamase
BBa_K627011
AraC_14 3C
BBa_K627013
TorA-TEV cleavage-β-lactamase
BBa_K627008
AraC_TEV
• Kill switch worked
TorA-TEV cleavage-β-lactamase
AraC_TEV
TorA-TEV cleavage-β-lactamase
0
20
40
60
80
100
120
0 25 50 75 100 150 200 300 400 600 800
Surv
ivin
g co
lon
ies
(%)
Ampicillin concentration in µg/ml
0 0 0 0 0 0 0
BBa_K627016
TEV
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In Vivo Selection
20
TorA
ampicillin
INHIBITION
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Modeling of in vivo Selection
Modeling: warum wieso?
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Based on the equations above, the kinetics of synthesis, export and interaction of the singel components can be modeled…
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Fitting the Model to Data
Model fitted to data
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The Progress Microcystis aeruginosa
mdn cluster
Library Generation
Selection System
Optimized Protease Inhibitor
![Page 24: Modification, Selection and Production of Cyclic Peptides2011.igem.org/files/presentation/Potsdam_Bioware_Championship.pdf · PowerPoint Presentation Author: iGEM11 Little Smurfs](https://reader036.vdocument.in/reader036/viewer/2022071015/5fce0a50cfae34408a33b099/html5/thumbnails/24.jpg)
24
Let‘s test the Libary - Results
Cells survive up to 400 µg/ml ampcillin
libary works
mdnA libary
+
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The Progress Microcystis aeruginosa
mdn cluster
Library Generation
Selection System
Optimized Protease Inhibitor
![Page 26: Modification, Selection and Production of Cyclic Peptides2011.igem.org/files/presentation/Potsdam_Bioware_Championship.pdf · PowerPoint Presentation Author: iGEM11 Little Smurfs](https://reader036.vdocument.in/reader036/viewer/2022071015/5fce0a50cfae34408a33b099/html5/thumbnails/26.jpg)
Human Practice and Xtras
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Parliament Survey
27
high medium low abstentionhigh medium
Synthetic Biology: Medicine and Healthcare
POTENTIAL RISK
Asked: >600 Answers: 10
Invitation to German
Parliament
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Visit the German Consulate
28
Boston, 4th November 2011
All German finalist iGEM-teams at the German Consulate in Boston (Bielefeld, Munich and Potsdam)
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Achievments and Future Plans
• achievments: – 13 BioBricks
• 5 for mdn modularization • 2 for phage display • 6 for in vivo selection
– proof of principle for all systems – talk with German parliamentarian
• future plans:
– using the libary against different proteases – inhibition studies for new Microviridins
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Achievments and Future Plans
… and another big achievment:
The BioLog App
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A(pp)tention Please…
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Thank you for your attention!
Dank: Instruktoren und Sponsore Refrences
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iGEM 2011 World Championship Jamboree at MIT in Boston
Sunday, 6th November 2011
Questions?