IGEM CHIBA 2009
The Blood Checkers
Fe Sensor ReporterHeme
Oxygenaze
Our Team
National University Corporation
Chiba University, JAPAN
(Member : )Kaoru YamamotoKazunao Hareyama Makoto NozawaSatoshi Ishizaki Yoshimi Iyama
Motivation
消化管の出血の有無を調査するのに最も簡単な方法として便潜血検査があります。便潜血検査には化学的方法と免疫学的方法の二種類あります。この二つの方法を組み合わせて、上部消化管からの出血は化学的方法で、下部消化管からの出血は免疫学的方法で検査をしています。ただし、化学的方法は疑陽性があることと、設備が揃っていない施設が多いため免疫学的方法だけで検査する場合が大半を占めるのが現状であり、大抵は上部消化管の検査ができていません。
私たちは上部消化管の胃の出血を検査するシステムをつくろうとしている。このシステムが完成すれば上部消化管の病気の早期発見に貢献するだろう。
System Overview
ReporterFe Sensor
HemeActivate
Heme oxygenase Generator
Biliverdin
1, Heme Oxygenage Generator
ReporterFe Sensor
Heme
Heme oxygenase Generator
Heme
Oxygenaze
Heme Oxygenage
Fe is contained in the Heme.To pick up Fe, we use this enzyme.
Do harm to organisms(←E. coliのほうがいいかも ?)
Express at (場所 )←どこでだろう…。未定（この有害さを抑える方法をこれから）
Breakdown of heme↓
Detection of Fe↓
Luminescence At the gut
Heme
Oxygenaze
Function of Heme Oxygenage
Heme + 3 AH2 + 3 O2 → biliverdin + Fe2+ + CO + 3A + 3 H2O
Heme BiliverdinFe
2+
Heme Oxygenage
Other
・ Catalyser of folowing reaction
Heme
Oxygenaze
Information of heme oxygenase
MERPQPDSMPQDLSEALKEATKEVHIQAENAEFMKNFQKGQVSREGFKLVMASLYHIYTALEEEIERNKQNPVYAPLYFPEELHRRAALEQDMAFWYGPHWQEIIPCTPATQHYVKRLHEVGRTHPELLVAHAYTRYLGDLSGGQVLKKIAQKAMALPSSGEGLAFFTFPNIDSPTKFKQLYRARMNTLEMTPEVKHRVTEEAKTAFLLNIELFEELQVMLTEEHKDQSPSQMASLRQRPASLVQDTAPAETPRGKPQISTSSSQTPLLQWVLTLSFLLATVAVGIYAM
Sequence
Sequence Length 289 aaMolecular Weight 32.9295 kDIsoelectric Point pH 6.08Extinction Coefficient 29990 M-1cm-1Absorption Coefficient 0.91Aliphatic Index 79.72
Heme
Oxygenaze
2, Fe Sensor
ReporterFe Sensor
Activate
Heme oxygenase Generator
Biliverdin
Fe Sensor
Fe PromoterFe Sensor
3, Reporter
ReporterFe Sensor
Activate
Heme oxygenase Generator
Biliverdin
Reporter
Sirius
What is Sirius?
Sirius is an ultramarine fluorescent protein.
What is amazing?
1.Brightness
2.pH-insensibility
3.The shortest fluorescent emission wavelength
Reporter
Brightness
Sirius is reported by Takeharu Nagai,a professor of nanosystem physiology at University of Hokkaido.
It has made from CFP which is a mutant of GFP.
Compared with Y66F mutant that made in the process,the brightness of Sirius is about 80 times of that of Y66F.
Also E.coli that cultured at 37℃ and that manifested Sirius showed 2 times of brightness of EBFP.
Thus,this fluorescent protein was named “Sirius” after the brightest and blue star in fixed stars.
Reporter
pH-insensibility
Thus far,quantities of light of fluorescent proteins became rapidly attenuate when pH is 7 or lower.
▼
Sirius’s quantities of light of fluorescent doesn’t change in condition of strong acid because of its pH-insensibility.
This can visualize two or more physiology phenomena in same cell.
By feed ameba bacteria which expressed Sirius,a series of process can be observed.
Upper:EGFP
Below:Sirius
Reporter
The shortest fluorescent emission wavelength
After the cloning is done,a lot of mutants of GFP were developed but the one with short wavelength was few.
One of the mutant that have short wavelength is BFP,which peak of absorb is 380 nm and peak of florescent is 450 nm.
Upper:A fluorescent spectrum of existing fluorescent protein and Sirius
Below:Refined,and excited these fluorescent protein
On the other hand,Sirius has 380 nm of peak of absorb and 424 nm of peak of florescent. Also,Sirius isn’t fade easily and its stability is about 60 times of BFP.
4, 胃とその他の区別Fe SensorHeme
Oxygenaze
ReporterFe SensorHeme oxygenase Generator
Biliverdin
pH sensor
ChannelChannel NhaANhaA
NhaRNhaANhaA
Activate
Na+ Na+H+ H+
BBa_K116001
When pH is higher, it senses the pH change and starts to work.
Fe SensorHeme
Oxygenaze
どのような実験やるか１、生理食塩水と Fe( )Ⅱ イオン水の混合液中に大腸菌を入れてみる（回路が機能しているか確かめる）
↓
２、擬似胃液、擬似腸液中でも回路が機能するか確かめる
↓
３、血液中で回路が機能するか確かめる
↓
完成？
得られそうなデータ・１の実験では鉄イオンが塩化物イオンと結合したり錯体形成したりする可能性がある。
・２の実験では胃液の酸性度で大腸菌が死んでしまうのではないか。
・３の実験ではうまく酵素を働かせられるか心配。
１の実験がうまくいけばシリウス（仮）が光る。
２の実験では多分シリウス（仮）は残って大腸菌は死んでしまうかもしれない。
３の実験はヘムを分解するのでだんだん入力が増えていって光ると思う。
Future Works
Acknowledgement
Prof. Daisuke Umeno
Prof. Taro Toyota
Prof. Kyoichi Saito
Hiroki Fukutomi
Masahiro Tominaga
Maiko Furubayashi
Biomaterial Laboratory Members
Ex – iGEMers
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