Team:Valencia/Parts

From 2009.igem.org

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<br>
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===Preparing inserts===
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=Our BioBricks=
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<br>
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We have contributed to the Registry with [http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2009&group=Valencia&Done=1 a set of parts], but one of them outstands all the others: [http://partsregistry.org/wiki/index.php?title=Part:BBa_K222000 aequorin]. This reporter protein needs a prosthetic group, coelenterazine, and citoplasmic Ca<sup>2+</sup> to work. This means that light is only emitted whenever the researcher uses coelenterazine and whenever there is a media full of Ca<sup>2+</sup>. There are some works that use aequorin to sense Ca<sup>2+</sup>, but we have tamed the protein to emit light whenever we want with the activation through electric current.
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Total DNA was extracted from our yeast strains.<br>
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A thorough characterization has been made and can be found either at the [http://2009.igem.org/Team:Valencia/Parts/Characterization corresponding page] in this wiki or at the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K222000 Registry].
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AEQ was amplified by PCR using oligonucleotides matching the sequence and bearing the appropriate Biobrick prefix and suffix.<br>
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Additionally, we have used a collection of knock outs in order to have genetic negative control that help us confirm that the light effect is not an artifact.
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Primers' sequences (EcoRI and XbaI sites in bold):<br>
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<br>
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Forward: 5' '''gAATTC'''gCggCCgCTTCTAgATggTgAgTTCgACAACTTC 3';<br>    
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<br>
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Reverse: 5' TACTAgTAgCggCCg'''CTgCAg'''CTATgTggTgCAgTCCACTg 3' <br>
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<center>[http://partsregistry.org/wiki/index.php?title=Part:BBa_K222000 http://2009.igem.org/wiki/images/3/35/Aequorin.GIF]
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<br>
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a 3D model of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K222000 aequorin], our outstanding BioBrick contribution to the Registry.
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PCR was conducted as follows:<br>
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</center>
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<ol>A first denaturation cycle
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<ol>94º 3'</ol>
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Followed by 30 amplification cycles:
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<ol>94º 30''
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60º 1' 30''
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72º 1'</ol>
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And a final extension step:
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<ol>72º 10'</ol>
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</ol>
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'''Results:'''<br>
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[[Image:.jpg]]<br>
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Intense amplicons, of the expected size, (about 1 kb) were produced for UCP-1, 175-deleted and 76 deleted. <br>
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Amplicons were digested (H buffer) with EcoRI y PstI.<br>
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Latest revision as of 23:24, 21 October 2009



Our BioBricks


We have contributed to the Registry with a set of parts, but one of them outstands all the others: aequorin. This reporter protein needs a prosthetic group, coelenterazine, and citoplasmic Ca2+ to work. This means that light is only emitted whenever the researcher uses coelenterazine and whenever there is a media full of Ca2+. There are some works that use aequorin to sense Ca2+, but we have tamed the protein to emit light whenever we want with the activation through electric current.

A thorough characterization has been made and can be found either at the corresponding page in this wiki or at the Registry.

Additionally, we have used a collection of knock outs in order to have genetic negative control that help us confirm that the light effect is not an artifact.

Aequorin.GIF


a 3D model of aequorin, our outstanding BioBrick contribution to the Registry.