Team:Valencia/Parts

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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 [https://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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<br>
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Firstly, aequorin sequence is:<br>
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<br>
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<center>[http://partsregistry.org/wiki/index.php?title=Part:BBa_K222000 https://static.igem.org/mediawiki/2009/3/35/Aequorin.GIF]
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1 atgaccagcg accaatactc agtcaagctt acatcagact tcgacaaccc aagatggatt<br>
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<br>
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61 ggacgacaca agcatatgtt caatttcctt gatgtcaacc acaatggaaa aatctctctt<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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121 gacgagatgg tctacaaggc atctgatatt gtcatcaata accttggagc aacacctgag<br>
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</center>
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181 caagccaaac gacacaaaga tgctgtagga gccttcttcg gaggagctgg aatgaaatat<br>
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241 ggtgtggaaa ctgattggcc tgcatacatt gaaggatgga aaaaattggc tactgatgaa<br>
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301 ttggagaaat acgccaaaaa cgaaccaacg ctcatccgta tatggggtga tgctttgttt<br>
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361 gatatcgttg acaaagatca aaatggagct attacactgg atgaatggaa agcatacacc<br>
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421 aaagctgctg gtatcatcca atcatcagaa gattgcgagg aaacatccag agtgtgcgat<br>
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481 attgatgaaa gtggacaact cgatgttgat gagatgacaa gacaacattt aggattttgg<br>
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541 tacaccatgg atcctgcttg cgaaaagctc tacggtggag ctgtccccta a<br>
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And our oligos (EcoRI and XbaI sites in bold) were:<br>
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Forward: 5'gaattcgcggccgcttctagatgaccagcg 3’<br>
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Reverse: 5’tactagtagcggccgctgcagttaggggac 3’<br>
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PCR was conducted as follows:<br>
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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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55º 1'
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72º 1'</ol>
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And a final extension step:
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<ol>72º 7'</ol>
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</ol>
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'''Results:'''<br>
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[[Image:.jpg]]<br>
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1 = wt (1 microlitre)<br>
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2 = wt (2 microlitres)<br>
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3 = Cch1 (1 microlitre)<br>
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4 = Mid1 (1 microlitre)<br>
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5 = Negative Control<br>
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6 = Possitive Control<br>
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(We used the same MWM)<br>
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We used wt 1 microlitre of PCR amplification product (career 1) to build the AEQ BioBrick.<br>
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Amplicons were digested (H buffer) with EcoRI y XbaI.<br>
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Latest revision as of 23:24, 21 October 2009



Our BioBricks


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 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 [http://partsregistry.org/wiki/index.php?title=Part:BBa_K222000 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.

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K222000 Aequorin.GIF]


a 3D model of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K222000 aequorin], our outstanding BioBrick contribution to the Registry.

Retrieved from "http://2009.igem.org/Team:Valencia/Parts"