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IGEM CHIBA 2009 The Blood Checkers Fe Sensor Reporte r Heme Oxygenaze

The Blood Checkers

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The Blood Checkers. Heme Oxygenaze. Reporter. Fe Sensor. IGEM CHIBA 2009. Our Team. National University Corporation Chiba University, JAPAN. (Member : ) Kaoru Yamamoto Kazunao Hareyama Makoto Nozawa Satoshi 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。 - PowerPoint PPT Presentation

Text of The Blood Checkers

Page 1: The Blood Checkers

IGEM CHIBA   2009

The Blood Checkers

Fe Sensor ReporterHeme

Oxygenaze

Page 2: The Blood Checkers

Our Team

National University Corporation

Chiba University, JAPAN

(Member : )Kaoru YamamotoKazunao Hareyama Makoto NozawaSatoshi Ishizaki Yoshimi Iyama

Page 3: The Blood Checkers

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。

Page 4: The Blood Checkers

System Overview

ReporterFe Sensor

HemeActivate

Heme oxygenase Generator

Biliverdin

Page 5: The Blood Checkers

1, Heme Oxygenage Generator

ReporterFe Sensor

Heme

Heme oxygenase Generator

Heme

Oxygenaze

Page 6: The Blood Checkers

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

Page 7: The Blood Checkers

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

Page 8: The Blood Checkers

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

Page 9: The Blood Checkers

2, Fe Sensor

ReporterFe Sensor

Activate

Heme oxygenase Generator

Biliverdin

Fe Sensor

Page 10: The Blood Checkers

Fe PromoterFe Sensor

Page 11: The Blood Checkers

3, Reporter

ReporterFe Sensor

Activate

Heme oxygenase Generator

Biliverdin

Reporter

Page 12: The Blood Checkers

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

Page 13: The Blood Checkers

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

Page 14: The Blood Checkers

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

Page 15: The Blood Checkers

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.

Page 16: The Blood Checkers

4, 胃とその他の区別Fe SensorHeme

Oxygenaze

ReporterFe SensorHeme oxygenase Generator

Biliverdin

Page 17: The Blood Checkers

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

Page 18: The Blood Checkers

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?

Page 19: The Blood Checkers

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。

Page 20: The Blood Checkers

Future Works

Page 21: The Blood Checkers

Acknowledgement

Prof. Daisuke Umeno

Prof. Taro Toyota

Prof. Kyoichi Saito 

Hiroki Fukutomi

Masahiro Tominaga

Maiko Furubayashi    

Biomaterial Laboratory Members

Ex – iGEMers