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

  • IGEM CHIBA2009The Blood Checkers

  • Our TeamNational University Corporation

    Chiba University, JAPAN(Member : ) Kaoru YamamotoKazunao Hareyama Makoto NozawaSatoshi Ishizaki Yoshimi Iyama

  • Motivation

  • System OverviewReporterFe SensorHemeActivateHeme oxygenase GeneratorBiliverdin

  • 1, Heme Oxygenage GeneratorReporterFe SensorHemeHeme oxygenase Generator

  • Heme OxygenageFe is contained in the Heme.To pick up Fe, we use this enzyme.

    Do harm to organisms(E. coli?)Express at ()

    Breakdown of hemeDetection of FeLuminescenceAt the gut

  • Function of Heme Oxygenage

  • Information of heme oxygenaseMERPQPDSMPQDLSEALKEATKEVHIQAENAEFMKNFQKGQVSREGFKLVMASLYHIYTALEEEIERNKQNPVYAPLYFPEELHRRAALEQDMAFWYGPHWQEIIPCTPATQHYVKRLHEVGRTHPELLVAHAYTRYLGDLSGGQVLKKIAQKAMALPSSGEGLAFFTFPNIDSPTKFKQLYRARMNTLEMTPEVKHRVTEEAKTAFLLNIELFEELQVMLTEEHKDQSPSQMASLRQRPASLVQDTAPAETPRGKPQISTSSSQTPLLQWVLTLSFLLATVAVGIYAMSequenceSequence Length289 aaMolecular Weight32.9295 kDIsoelectric PointpH 6.08Extinction Coefficient29990 M-1cm-1Absorption Coefficient0.91Aliphatic Index79.72

  • 2, Fe SensorReporterFe SensorActivateHeme oxygenase GeneratorBiliverdin

  • Fe Promoter

  • 3, ReporterReporterFe SensorActivateHeme oxygenase GeneratorBiliverdin

  • SiriusWhat is Sirius?Sirius is an ultramarine fluorescent protein.What is amazing?1.Brightness2.pH-insensibility3.The shortest fluorescent emission wavelength

  • BrightnessSirius 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.

  • pH-insensibilityThus far,quantities of light of fluorescent proteins became rapidly attenuate when pH is 7 or lower.Siriuss quantities of light of fluorescent doesnt 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:EGFPBelow:Sirius

  • 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 SiriusBelow:Refined,and excited these fluorescent proteinOn the other hand,Sirius has 380 nm of peak of absorb and 424 nm of peak of florescent. Also,Sirius isnt fade easily and its stability is about 60 times of BFP.

  • 4, ReporterFe SensorHeme oxygenase GeneratorBiliverdin

  • pH sensorChannelNhaANhaAActivateNa+Na+H+H+BBa_K116001

    When pH is higher, it senses the pH change and starts to work.

  • Fe()

  • Future Works

  • AcknowledgementProf. Daisuke UmenoProf. Taro Toyota Prof. Kyoichi SaitoHiroki FukutomiMasahiro TominagaMaiko Furubayashi Biomaterial Laboratory MembersEx iGEMers

  • Future WorksGFPYFPPrFeJ3902PrFeJ3902PnhaAK116001PcpH7pH sensor -> not ActivePrFeYFPPrFeGFP pH7pH sensorActivePrFeYFPPrFeGFP

    This is our team members.

    We have 5 undergraduate students. 4 of us major chemistry, and 1 major biology.

    *We want to make the system that detects the hemorrhage in the gut. First, we target the iron contained in the hemoglobin. Our system break down the hemoglobin by heme oxygenase, and iron is taken out. Then PrFe reacts to the iron and it informs us of the existence of iron by expressing of repoerter protein. Second, the pH environment is not constant according to the place in the gut. So we will make parts that function as a reporter even if the pH environment changes.

    *ph

    *5591 45%96%http://www.jrcs-kanagawa.org/ketueki/blood/knowledge/component.html97%BiobrickFeSensorFe

    Heme oxygenase GeneratorHemeHeme oxygenase*Fe*FeFe

    Fe*pH

    **

    *GFPY66F37EBFP*pH7pHpH3EGFP*GFP

    pH**(II) FeCl2(III) FeCl3 [FeCl4]2 4 *