modification, selection and production of cyclic...

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


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


mdn cluster

Library Generation

Selection System


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


AraC_TEV


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


mdn cluster

Library Generation

Selection System


24

Let‘s test the Libary - Results

Cells survive up to 400 µg/ml ampcillin

libary works

mdnA libary

+


mdn cluster

Library Generation

Selection System


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?

modification, selection and production of cyclic...

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