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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
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!
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