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The Blood Checkers

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。

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The Blood Checkers

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  1. The Blood Checkers Heme Oxygenaze Reporter Fe Sensor IGEM CHIBA2009

  2. Our Team National University Corporation Chiba University, JAPAN (Member : )Kaoru Yamamoto Kazunao Hareyama Makoto Nozawa Satoshi Ishizaki Yoshimi Iyama

  3. 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。

  4. System Overview Heme Biliverdin Activate Heme oxygenase Generator Reporter Fe Sensor

  5. 1, Heme Oxygenage Generator Heme Heme Oxygenaze Heme oxygenase Generator Reporter Fe Sensor

  6. 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） Heme Oxygenage Heme Oxygenaze Breakdown of heme ↓ Detection of Fe ↓ Luminescence At the gut

  7. ・Catalyser of folowing reaction Heme + 3 AH2 + 3 O2 → biliverdin + Fe2+ + CO + 3A + 3 H2O Other Heme Oxygenage Biliverdin Heme 2+ Fe Function of Heme Oxygenage Heme Oxygenaze

  8. Information of heme oxygenase Sequence Length 289 aa Molecular Weight 32.9295 kD Isoelectric Point pH 6.08 Extinction Coefficient 29990 M-1cm-1 Absorption Coefficient 0.91 Aliphatic Index 79.72 Heme Oxygenaze MERPQPDSMPQDLSEALKEATKEVHIQAENAEFMKNFQKGQVSREGFKLVMASLYHIYTALEEEIERNKQ NPVYAPLYFPEELHRRAALEQDMAFWYGPHWQEIIPCTPATQHYVKRLHEVGRTHPELLVAHAYTRYLGD LSGGQVLKKIAQKAMALPSSGEGLAFFTFPNIDSPTKFKQLYRARMNTLEMTPEVKHRVTEEAKTAFLLN IELFEELQVMLTEEHKDQSPSQMASLRQRPASLVQDTAPAETPRGKPQISTSSSQTPLLQWVLTLSFLLA TVAVGIYAM Sequence

  9. 2, Fe Sensor Biliverdin Activate Fe Sensor Heme oxygenase Generator Reporter Fe Sensor

  10. Fe Promoter Fe Sensor

  11. 3, Reporter Biliverdin Activate Reporter Heme oxygenase Generator Reporter Fe Sensor

  12. What is Sirius? Sirius Reporter Sirius is an ultramarine fluorescent protein. What is amazing? 1.Brightness 2.pH-insensibility 3.The shortest fluorescent emission wavelength

  13. 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

  14. 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. Reporter By feed ameba bacteria which expressed Sirius,a series of process can be observed. Upper:EGFP Below:Sirius

  15. 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. 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. Upper:A fluorescent spectrum of existing fluorescent protein and Sirius Below:Refined,and excited these fluorescent protein

  16. 4, 胃とその他の区別 Biliverdin Heme Oxygenaze Fe Sensor Heme oxygenase Generator Reporter Fe Sensor

  17. pH sensor Na+ H+ Na+ H+ BBa_K116001 When pH is higher, it senses the pH change and starts to work. Heme Oxygenaze Channel NhaA Fe Sensor Activate NhaR NhaA

  18. 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？

  19. 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。

  20. Future Works

  21. 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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