Post on 22-Aug-2020
iGEM 2011 World Championship Jamboree at MIT in Boston
Sunday, 6th November 2011
Modification, Selection and Production of Cyclic Peptides
Outline
Laboratory
Introduction
Outreach & Xtras
2
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
4
Microviridin Family
• cyclization over side chain residues – no cysteins or any backbone structure
involved
• cyclic peptide with two ester and one amide bond
The Project Microcystis aeruginosa
mdn cluster
Library Generation
Selection System
Optimized Protease Inhibitor
Heterologous Expression in E. coli
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
Characterization
7
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
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
The Progress Microcystis aeruginosa
mdn cluster
Library Generation
Selection System
Optimized Protease Inhibitor
coating
Phage Display
10
M13 Phage
Pili
mdnA-myc-geneIII
binding
Infected E.coli
washing
washing
elution infect E.coli
11
Preparing the Phage
mdnA-myc-tag-geneIII
M13 Phage
gene III microviridin
myc-tag
mdnC mdnB mdnA-myc-geneIII mdnD mdnE
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
Test Panning
13
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
14
Panning with Different Proteases
0
20
40
60
80
100
120
clon
e nu
mb
er a
fter
pa
nnin
g
• microvirdin binds papain
The Progress Microcystis aeruginosa
mdn cluster
Library Generation
Selection System
Optimized Protease Inhibitor
In Vivo Selection
16
ssTorA
• Basic Idea • β–lactamase is only active in
periplasm • signal sequence of TorA serves
as a export signal
ampicillin
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
In Vivo Selection
18
proteolytic cleavage • cells will die
ampicillin
protease
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
In Vivo Selection
20
TorA
ampicillin
INHIBITION
Modeling of in vivo Selection
Modeling: warum wieso?
21
Based on the equations above, the kinetics of synthesis, export and interaction of the singel components can be modeled…
22
Fitting the Model to Data
Model fitted to data
The Progress Microcystis aeruginosa
mdn cluster
Library Generation
Selection System
Optimized Protease Inhibitor
24
Let‘s test the Libary - Results
Cells survive up to 400 µg/ml ampcillin
libary works
mdnA libary
+
The Progress Microcystis aeruginosa
mdn cluster
Library Generation
Selection System
Optimized Protease Inhibitor
Human Practice and Xtras
26
Parliament Survey
27
high medium low abstentionhigh medium
Synthetic Biology: Medicine and Healthcare
POTENTIAL RISK
Asked: >600 Answers: 10
Invitation to German
Parliament
Visit the German Consulate
28
Boston, 4th November 2011
All German finalist iGEM-teams at the German Consulate in Boston (Bielefeld, Munich and Potsdam)
29
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
30
Achievments and Future Plans
… and another big achievment:
The BioLog App
31
A(pp)tention Please…
32
Thank you for your attention!
Dank: Instruktoren und Sponsore Refrences
iGEM 2011 World Championship Jamboree at MIT in Boston
Sunday, 6th November 2011
Questions?