Project overview

Project overview

Aggregation using Ag43

EASY

DIFFICULT

Ag43

Project overview

The best way to produce Bio-plastic

Project overview

Aggregation Module Plastic Producing Module

Hard Cluster Bio capsule

Valuable substance

Harvesting by Filtration＆ Decantation

Bio capsule

Project overview

Biobrick Review

• A bio-communication tool for iGEMers.

• You can learn more biobrick parts and communicate with other iGEMers

all over the world.

Centrifugation takes much time

Centrifuge

Culture Medium

Remove Supernatant

Add Medium

Filtration can harvest continuously

Filtration

Continuous supply of medium

Filtrate Culture Medium

Antigen 43 (Ag43) is…

• An adhesion molecule

• A cell surface protein

• Assists aggregation

Ag43 expression image

Signal peptides

α domain

Autochaperone

β domain

Outer Membrane

Inner Membrane

Ag43 Complex

α

α α

β β β

Induction of aggregation module

Translation

AraC

L-arabinose

PBAD RBS dT Ag43

AraC

Aggregation was confirmed

• 5ml LB medium

• Rotary shaking

at 180 rpm, 37℃

• Medium is clear in the

presence of arabinose Arabinose(-) Arabinose(+)

Aggregation result in reduction of optical density

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 2 4 6 8 10 12 14

⊿O

D6

00

of

me

diu

m

Time (hours)

Arabinose(+)

Arabinose(-)

Arabinose(-) Arabinose(+)

Harvesting method Stroking

Filtrate

Mesh

Culture Medium

Example of filtration

Arabinose(-) Arabinose(+)

36 hrs cultivation (5 ml LB) mesh size: 250 um

Summary

• Characterization our aggregation module

• Establishment of an efficient method for aggregating cells to a cluster

Submitted parts

p B A D +

A r a C R B S e C F P R B S A g 4 3 d T

B B a _ I 0 0 5 0

B B a _ B 0 0 3 4

B B a _ E 0 0 2 0 B B a _ K 3 4 6 0 0 7

B B a _ B 0 0 1 5 B B a _ B 0 0 3 4

p B A D +

A r a C R B S A g 4 3 d T

B B a _ I 0 0 5 0

B B a _ B 0 0 3 4

B B a _ K 3 4 6 0 0 7

B B a _ B 0 0 1 5

What is Poly(3HB) ?

Poly-3-hydroxy-butyrate, Poly(3HB) is bio-plastic.

It is made by bacteria as an energy-storage molecule. → bio-degradable

It is made from glucose or soybean oil. It is a part of carbon cycle.

P(3HB) is made from acetyl-CoA in 3steps

Three enzymes encoded in phaCAB operon works to produce bio-plastic.

Product identified as Poly(3HB) with GC/ MS.

▲ standard

Products by pGEM ▼ Peak comparison

We quantitatively analyzed Poly(3HB) production among various conditions

Transformed plasmid: pGEM(PhaCAB)

TB is the best medium to produce Poly(3HB) !

Best condition for Poly(3HB) production

TB helps bacteria growth and Poly(3HB) production.

Pantothenic acid and Glucose are substrates of the pathway.

JM109 is more closely related to wild-type than DH5α.

We successfully identified and quantified Poly(3HB)!

We found a substitute for common plastic

and produce it in E.coli.

We identified the product as bio-plastic, Poly(3HB).

We established the best condition to produce bio-plastic.

Bio capsule system

Plastic producing module Aggregation

module

Experiments

• We transformed a plasmid of “plastic producing module” into the cell of “Aggregation module”

• The “Aggregation module” is induced by the addition of 1% Arabinose.

• The “Plastic producing module” is constitutively expressed and supplemented with addition of glucose.

Stronger aggregation produced clearer supernatant

Ara Glu

(+) (+)

(-) (+)

24 hrs

48 hrs

Conditions 37℃, 180 rpm, 1.5 ml

(+) (-)

Clusters of cells

Fast precipitation was observed

After incubation of 48 hrs, 180 rpm, 50 ml

With both modules precipitation was even faster!

0 min

5 min

10 min

Ara Glu

(+) (-)

(+) (+)

(-) (+)

Aggregation module Plastic producing module

(+) (-)

(+) (+)

Nile red staining

Bright field

The production of P3HB was confirmed

Arabinose induction Glucose induction

The production of P3HB was confirmed

<HPLC test>

(+) (+) (-) (-) (+) (+)

We can collect cells by filtration or by decantation in large scales !

The expression plastic increases the strength of aggregation!

We submitted eight parts

RBS PhaC RBS PhaA RBS PhaB dT

B0034 K342001 K759004 K759005 B0015

K759006 K759007 K759008

K759009

K759010

B0034 B0034

We are now testing the production of plastics with our parts!

Contribution to manufacturing

• Bio capsule system largely contributes to the method of harvesting bacterial cells!

• The model system for producing P3HB can be extended to other kinds of plastic production!

Easy and low costing!

Human Practice

A Brand New Bio-communication Tool

Our Issues

Too detailed information in PartsRegistry

Unexpected behaviors of BioBrick Parts

Solution

We want to share OUR opinions.

“Biobrick Review”

Discuss issues of BioBricks!

“Biobrick Review”

Learn characters of BioBricks!

Enjoy Ranking Events as GAMEs!

How to Use BB Review

Visit http://www.bbreview.net

How to Use BB Review

Get your account

How to Use BB Review

Find a BioBrick

How to Use BB Review

Post your review!!

For Development

WE NEED

YOUR

PARTICIPATION

Enjoy www.bbreview.net

RIGHT NOW!

Achievements Submitted and characterized 11 biobricks 2 biobricks for aggregation module

9 biobricks for bio-plastic producing module

Succeeded in making “Bio capsule” by combining aggregation protein expression and bio-plastic synthesis

released biobrick social site “biobrick review” to evaluate each biobrick through enjoying ranking like a game

Participated in all Japan pre-jamboree convention

Had a boot camp for beginners education

Found a few faulty registered parts and improved them

Succeeded in synthesizing bio-plastic and confirming the product by GC/MS

Had lots of fun Threw a barbeque, go skiing, and having some parties!!!!!

References

• van der Woude, Marjan W Henderson, Ian R. Regulation and function of Ag43 (flu). Annu. Rev. Microbiol. 2008. 62:153–69 http://www.ncbi.nlm.nih.gov/pubmed/18785838

• P Caffrey and P Owen. Purification and N-terminal sequence of the alpha subunit of antigen 43, a

unique protein complex associated with the outer membrane of Escherichia coli. J. Bacteriol. 1989, 171(7):3634. http://www.ncbi.nlm.nih.gov/pubmed/2661530

• Inga Benz, M. Alexander Schmidt. Structures and functions of autotransporter proteins in microbial pathogens. International Journal of Medical Microbiology 301 (2011) 461– 468. http://www.ncbi.nlm.nih.gov/pubmed/21616712

• Glen C. Ulett, Jaione Valle, Christophe Beloin, Orla Sherlock, Jean-Marc Ghigo and Mark A. Schembri. Functional Analysis of Antigen 43 in Uropathogenic Escherichia coli Reveals a Role in Long-Term Persistence in the Urinary Tract Infect. Immun. 2007, 75(7):3233. DOI: 10.1128/IAI.01952-06. http://iai.asm.org/content/75/7/3233.abstract

• http://2010.igem.org/Team:Peking/Project/Bioabsorbent/InductiveAggregation

• http://openwetware.org/wiki/Arking:JCAOligoTutorial8

• http://www.jcat.de/

Aggregation module

References

• H.Ohara. Change from Oil-based to Bio-based. Sen’i gakkaishi 66, 4.129-132 (2010)

• 2. Pohlmann, A. et al. Genome sequence of the bioplastic-producing “Knallgas” bacterium Ralstonia eutropha H16. Nature biotechnology 24, 1257–62 (2006).

• 3. 田口精一: バイオプラスチックのつくられ方とつくり方 蛋白質 核酸 酵素，50.3: 262-269 (2005) (in Japanese)

Bio-plastic synthesis

Acknowledgements

• Yamazaki Lab, Environmental Molecular Biology, Graduate School of Environmental Earth Science, Hokkaido University

• Taguchi Lab, BioMolecular Engineering, Graduate School of Enggineering, Hokkaido University

Sponsors

Thanks for listening!