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 [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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<br>
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<br>
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And our oligos (EcoRI and XbaI sites in bold) were:<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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Forward: 5'gaattcgcggccgcttctagatgaccagcgaccaatactc 3’<br>
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<br>
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Reverse: 5’tactagtagcggccgctgcagttaggggacagctccaccg 3’<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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</center>
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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 3min</ol>
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Followed by 30 amplification cycles:
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<ol>94º 30s<br>
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55º 1min<br>
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72º 1min<br></ol>
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And a final extension step:
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<ol>72º 7min</ol>
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</ol>
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[[Image:Gelaeq.JPG]]
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'''Results:'''<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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Amplicon has 600 pb's.
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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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===Preparing vectors===
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Competent cells were transformed with: <br>
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Plasmids were extracted (High pure miniprep plasmid isolation kit ROCHE) <br>
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Plasmid were digested with EcoRI and XbaI<br>
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===Ligating Biobricks into plasmids===
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Both plasmids and inserts were run into 0.8% 0.5X TBE agarose gels and DNA bands excised with a clean scalpel. DNA was extracted from agarose blocks (ultra clean gel spin, DNA purification Kit, MO BIO laboratories).<br>
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T4 Ligase was used to ligate inserts and vectors for 1 h at room temperature (2X quick buffer was used).<br>
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Competent cells were transformed and resulting colonies (Amp LB) screened with Fw and Rv primers to confirm the presence of inserts. <br>
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pSB1AK3 containing UCP-1, 175-deleted and 76-deleted were sent to the Registry <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.