nymu-taipei 2011 igem team2011.igem.org/files/presentation/nymu-taipei.pdf · lo a., chiu y.y.,...

NYMU-Taipei 2011 iGEM Team Taiwan National Yang Ming University

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Tailoring Your

Avatar

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Mind-Transferring Machine

To realize the Avatar fantasy !

National Yang Ming UniversityNational Yang Ming University

Remote Modulation of Neurons

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Neuron Modulation

Optogenetics

<Nature Method> Method of the Year 2010

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nerve cell


Remote Modulation

Gravity

Weak Nuclear force

Strong Nuclear force

Magnetic Force Electro -magnetism

Magnetotactic Bacterium 6


Our General Design

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glial cell

National Yang Ming University

Symbiosis between magnetotactic bacterium and glial cell


Our General Design

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nerve cells

magnetotactic baterium in glial cell


16 / -Some have listed specific aims for their magneto-project proposals; -All given clear mentions to “magnetic bacteria/magnetotaxis” on official wikis. YET, FOR TEAMS THAT REALLY GOT STARTED WITH MAGNETIC BACTERIA…

2004 2005 2006 2007 2008 2009 2010 2011

2011 Washington 2008 Cambridge

2011 Cambridge

2008 TU Delft

2011 Toronto

2009 Cornell

2010 Northwestern

2006 Brown

2006 Duke

History

2011 UPenn

2009 Cornell

2010 TU Delft

2010 Uppsala Sweden

2011 NYMU-Taipei

year

2007 Uppsala Sweden

2006 MIT

Bacterial Dynamo

: AC generator with magnetic bacteria circulating a center

Dr

ea

m c

ha

ss

is

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Magnetospirillum magneticum strain: AMB-1

Gram- negative Non-pathogenic

Aquatic, Motile

magnetosomes


Magnetotactic Bacteria

Shuttle Vector pYMB

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GFP emission

light intensity

(unit)

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pYMB expressing GFP Competence & Efficacy Confirmation

Signal

Transcription

Translation

Protein Modification

Functional Protein

Biochemical Pathway

Biophysical Pathway

Signal

Simultaneous Response

How to Enlighten the Bacteria?


magnetite

membrane

Mm

s13(H2)

Mm

s13(H1)

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The Magnetotactic Bacterium AMB-1

The Model Imaging of Magnetosomes in AMB-1

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 2006, p. 465–471 Vol. 72, No. 1

Inspiration of Our Design

mms13


H1

Rluc

H2

YFP

H1

Mms13 in off state

• When a magnetic field is applied: – The magnet thing is

pulled – Alpha helices H1 and H2

of Mms13 move together

Rluc

H2

YFP

OFF ON

Design

【BRET】 Bioluminescence

Resonance Energy Transfer


H1

Mms13 in on state • When a magnetic field is applied:

– The magnet thing is pulled – Alpha helices H1 and H2 of

Mms13 move together

• When YFP is close to Rluc, it induces the BRET phenomenon and YFP lights up

• Problem: It is ALWAYS on the on state

─ Due to tight interaction within two helices

Rluc

H2

YFP

OFF ON

Design National Yang Ming University

Rluc

H2 CH

AMP

H1

Solution? Insert CHAMP protein

• Insert the CHAMP protein!

• Designed to fit with H1 with a higher affinity than the H2 helix

Rluc

H2 CH

AMP

H1

Split YFP

OFF ON

Design

【BiFC】-based BRET Bimolecular Fluorescence Complementation-based

BRET CHAMP

Computed helical anti-membrane protein


Lighting up again!

• The on state (magnetized) Rluc

H2 H1

Split YFP

CH

AMP

OFF ON


Turning off

• Turning off now works since H2 is not attached to H1

Rluc

H2 CH

AMP

H1

Rluc

H2 CH

AMP

H1

Split YFP

OFF ON


• Choose the template pairs with one helix has most affinity and the other helix most likely to helix 1 of Mms13.

CHAMP Design

Science 315, 1817 (2007) Computational Design of Peptides That Target Transmembrane Helices Hang Yin, et al.

PNAS September 12, 2006 Helix-packing motifs in membrane proteins R. F. S. Walters, W. F. DeGrado

Bioinformatics (2009) 25: 996-1003. Predicting helix-helix interactions from residue contacts in membrane Lo A., Chiu Y.Y., Rødland E.A., Lyu P.C., Sung T.Y., and Hsu W.L.

1. Selection of templates for Computed helical anti-membrane protein (CHAMP) design

2. Packing the side chains to minimize the total energy of the complex structure

Modeling

• 445 natural helical pairs were clustered into groups based on their three-dimensional similarity.

CH

AM

P

Target helix G

-X3-G

• G-X3-G motifs have a flat helix surface which can maximizes van der Waal’s interactions and the loss of side-chain entropy

• Gly residues in the motifs also reduce the distance between the helix axes

CH

AMP

Target helix G

-X3-G


CHAMP Design




2. Packing the side chain to minimize the total energy of the complex structure

Modeling

Etotal = EvdW

+Ehbond

+Eelectrostatic

+Esolvation

+Eself

+Econtact


G-X3-G

Result for Cross-model Majority Vote

Helix 1 ⇒ 12~29

Helix 2 ⇒ 65~87

sosui DAS(low cut-off)

DAS(high cut-off)

TMHMM PredictProtein TMpred

HMMTOP MEMSAT-SVM TopPred

Mms13 Targeted by CHAMP



Bioinformatics (2009) 25: 996-1003. Predicting helix-helix interactions from residue contacts in membrane Lo A., Chiu Y.Y., Rødland E.A., Lyu P.C., Sung T.Y., and Hsu W.L.

Selection of templates for Computed helical anti- membrane protein (CHAMP) Design

Result of Modeling National Yang Ming University



Result of Modeling

Packing the side chain to minimize the total energy of the complex structure

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-0.10

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G A V I L R K H W Y F N D Q E S T C M P

End score calculated by YASARA Energy Minimization Server

End

sco

re

calc

ulat

ed

Predicted Δ

Gapp

(kcal mol -1)

-0.6

-0.4

-0.2

1E-15

0.2

0.4

0.6


End score calculated by YASARA Energy Minimization Server

End

sco

re

calc

ulat

ed

Predicted Δ

Gapp

(kcal mol -1)

Mms13 Targeted by CHAMP

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0.1

0.2

0.3

0.4

0.5

0.6


End

scor

e

calc

ulat

ed

Predicted ΔGapp (kcal m

ol -1)

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0.90

1.00


End

scor

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calc

ulat

ed


ol -1)

-1.20

-1.00

-0.80

-0.60

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0.60

0.80

1.00

End

scor

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calc

ulat

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ol -1)

-0.35

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0.55

0.7

0.85

1

End

scor

e

calc

ulat

ed


ol -1)

-0.80

-0.60

-0.40

-0.20

0.00

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0.60

0.80

End

scor

e

calc

ulat

ed


ol -1)


2. Packing the side chain to minimize the total energy of the complex structure

Final choice Final choice

Final choice Final choice Final choice

Final choice


Using BRET to test whether YFP anchored in N-terminus of Mms13 will effect gene expression. Using CHAMP to test the on-off system. The ideal construct. Using BiFC-based BRET to

improve H1 and CHAMP’s affinity.

Constructs

Rluc

H2 CH

AMP

H1

Rluc H2 H1

Rluc

H2

CH

AMP

H1

YFP

Rluc

H2 CH

AMP

H1

YFP

Rluc

H2 CH

AMP

H1

Rluc

H2 H1

YFP

Positive Control with rLuc anchored on Mms13


• We need the gradient about 8.176*105 T/m • One little magnet can only apply force up to 900~1000G

Modeling For Optomagnetic Design

• It is known that 2 nN applied to the cell will cause large distortion on the cell

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Biophysical Journal Volume 85 July 2003 623–636 623 Tensile Force-Dependent Neurite Elicitation via Anti-b1 Integrin Antibody-Coated Magnetic Beads Joseph N. Fass* and David J. Oddey

Am J Physiol Cell Physiol 287: C1–C11, 2004; 10.1152/ajpcell.00559.2003. Cell mechanics and mechanotransduction: pathways, probes, and physiology Hayden Huang,1 Roger D. Kamm,2 and Richard T. Lee1,2

• The applied gradient is about 8.176*105 T/m

• If tested the electric Transformer box’s magnetic gradient away from one meter in radius and one meter for height, the gradient is about 200 mG


• What will be the responses following the injection of AMB-1?

• What if AMB-1 grows in brain without control? • How does AMB-1 stay within?

Rescuing the brain From detrimental inflammation


Establishing Symbiosis with brain… Using Extra- or Intra- cellular strategy? 1. Why not extracellular? • This generates difficulty and uncertainty to prevent AMB-1

from fleeing the injected site, which undermines accuracy.

2. Reviewing the past researches… • Most bacteria infectious to brain replicate intracellularly.

e.g. Yersinia enterocolitica Listeria Monocytogenes Mycobacteria tuberculosis

We chose to establish intracellularity!!!


Cells we targeted for NeuroSymbiosis 1. Neurons • May automatically trigger neuronal activity when introducing

AMB-1 into neurons.

2. Glial cells • Focus on the relationship between AMB-1 & glial cells!!

We chose glial cells as our neurosymbiosis target!



1. Invasin BBa_K177010 • A gene that promotes bacterial entry to glial cell

2. LLO BBa_K177026 • A gene that facilitates bacterial escape from phagosomes

3. MinC BBa_K624033 • A cell division inhibitor • Upload coding sequence 2006 Chiba • Provide physical DNA 2010 Warsaw • Remove illegal PstI site with one-step PCR 2011 NYMU-Taipei

“AMB-1tious” solution for NeuroSymbiosis

Glial cell

B-1 Integrin Receptor





3. MinC BBa_K624033 • A cell division inhibitor • Upload coding sequence 2006 Chiba • Provide physical DNA 2010 Warsaw • Remove illegal PstI site with one-step PCR 2011 NYMU-Taipei


x





3. MinC BBa_K624033 • A cell division inhibitor • Merely uploaded sequence 2006 Chiba team • Provide physical DNA with PstI (illegal) 2010 Warsaw team • Remove illegal PstI site with one-step PCR 2011 NYMU-Taipei


We have IMPROVED the part and followed the standards!!! BBa_K624033





3. MinC BBa_K624033 • A cell division inhibitor


invasin 2508bp

3000bp

LLO 1590bp

1500bp

minC 696bp

600bp 700bp minC 696bp

700bp

Genomic PCR cloned from Yersinia enterocolitica

Genomic PCR cloned from Listeria Monocytogenes

Genomic PCR cloned from E. coli K12



Two constructs made for NeuroSymbiosis 1. Tetracycline-induced system on AMB-1 to regulate minC

expression

2. We referenced 2010 Warsaw team’s work and designed and made our own constructs of Invasin and LLO.


72 kDa

26 kDa

Supernatant Control

Supernatant Experiment

Pellet Control

Pellet Experiment

• The expression of TetR protein enabled our regulation.

GFP 27 kDa

TetR 24 kDa


Two constructs made for NeuroSymbiosis 1. Tetracycline-induced system on AMB-1 to regulate minC

expression

2. We referenced 2010 Warsaw team’s work and designed and made our own constructs of Invasin and LLO.


Glial cells AMB-1


Applications

Intracellular NeuroSymbiosis


Online Primer Software

Bbpprimer—Davidson college

PCR primer design tool—Ginkgo Biowork

ATCGGTAGGATCGATGCATGCTAGCATCGATCGTAGGATCGTAGCTAGCGATCGATGCATGATCGATCGTAGCTATCTATGCTAGCTAGCTATCGTCTAGCT

ATCGGTAGGATCGATGCATGCTAGCATCGATCGTAGGATCGTAGCTAGCGATCGATGCATGATCGATCGTAGCTATCTATGCTAGCTAGCTAGCTAGCTAGCT

1. Sequence resource not available 2. Primer lacking analysis


Features

1. Extracting 1400 total genome, including AMB-1 2. Quantitative analysis for primers Prefix Suffix


Human Practice

(1) Opening Survey (2) Discussion (3) Closing Survey

New Approach:

71% highly interested

22% interested

7% less interested


Roles Viewpoints Physician Healing Parkinson’s disease and Alzheimer’s disease Educator Strengthen memory, creating more Einstein! Police Control behaviors of outrageous criminals. Law maker Increasing difficulties for making regulations. Taxonomist New strategies should be applied to new species.

Role-Playing Strategy

Human Practice (1) Opening Survey (2) Discussion (3) Closing Survey

New Approach:

1. Neuroethics 2. Patent in Synthetic fields 3. Synthetic Neurobiology


52 52

Human Practice

52

(1) Opening Survey (2) Discussion (3) Closing Survey

New Approach:

21% Theme well-discussed

10% No opinion

69% Neuroethics requires further attention from society


International Collaboration

• We collaborate with China teams on establishing chassis “AMB-1”

Tsinghua Tianjin UST-Beijing

AMB-1 growth curve


O eurOsynbiOsis

PtOmagnetic twinkles

n agnetic M chassis

Designed a de novo vital inhibitor peptide,

CHAMP, with 3 full PCR-constructs.

Made outstanding modeling to prove the concept of magnetosome-driven optogenetic stimulation.

Successfully cloned, constructed, and submitted 3 crucial parts for optomagnetic twinkling.

Given a unprecedented one-step-in vivo design for prokaryote-based treatment

Successfully cloned, submitted and removed illegal sites from 3 essential symbiotic parts.

Hosted 1 whole-campus convention and

7 visits, expanding the realm of synthetic biology,

1 role-playing event to investigate neuroethical scenarios.

uman H Practice

ollaboration C &

Readily cloned 3 strongest

promoters and 2 rbs,

Designed, constructed and fully characterized a shuttle

vector, pYMB, in

collaboration with 3 teams.

date

~

~

achievements

cultivation of AMB-1

Chassis set-up, Constructs completed

Expressing constructs on E. coli and AMB-1; conducting

thorough tests on all hypotheses

Co-culture with light-sensitive neurons

Scientific Significance achieved

Scientific Impact Given

2D Optogenetic neural network activation

Nir Grossman, Patrick Degenaar, et al., 2010, J. Neural Eng. , 7, 016004

Wireless, Multichannel neural amplifier Nir Grossman, Patrick Degenaar, et al., Nat. Neurosci., Vol. 4 (2), pp263-270 (2011)

Mind-altering Bacteria Greg Miller, Mind-Altering Bugs, Science Now, 29 August 2011

Designed a de novo vital inhibitor peptide, CHAMP, with 3 full PCR-constructs.

Made outstanding modeling to prove the concept of magnetosome-driven optogenetic stimulation.

Successfully cloned, constructed, and submitted 3 crucial parts for optomagnetic twinkling.

Given a unprecedented one-step-in vivo design for prokaryote-based treatment

Cloned and submitted 3 strongest promoters and 2 rbs,

Designed, constructed and fully characterized a shuttle vector, pYMB, in collaboration with 3 teams.

Hosted 1 whole-campus convention and 7 visits, expanding the realm of synthetic biology,

1 role-playing event to investigate neuroethical scenarios.

Thanks for your listening

achi

evem

ents

Dr. Nien-Jung Chen Dr. Hsei-Wei Wang

Dr. May-Ru Chen Dr. Cheng-Chang Lien Dr. Wailap Victor Ng

Dr. Ueng-Cheng Yang

Dr. Ming Cheng

Dr. Han-Juo Cheng

Dr. An-Na Chiang

Dr. Sheh-Yi Sheu

Dr. Jyun-Min Luo

Chancellor Dr. Kung-Yee Liang

A c k n o w l e d g e m e n t s :

S p o n s o r e d b y :

nymu-taipei 2011 igem team2011.igem.org/files/presentation/nymu-taipei.pdf · lo a., chiu y.y.,...

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