Wiki/Notebook wetlab.html

From 2014.igem.org

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<h3><p></p>Biosynthesis</h3>
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</br></br><h3><p></p>Biosynthesis</h3>
 +
 
 +
</br><p><h4>06/09/2014</h4></p>
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<p><h4>06/09/2014</h4></p>
 
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<p>
 
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</p>
 
<p>The enzymes involved in the biosynthesis pathways are AtrD11, HarFAR, FAO, EaDAcT.
<p>The enzymes involved in the biosynthesis pathways are AtrD11, HarFAR, FAO, EaDAcT.
</p>
</p>
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<p>
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-
</p>
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<ul><li>Codon optimization
<ul><li>Codon optimization
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</ul>
</ul>
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<p>
 
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</p>
 
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<p><h4>06/10/2014</h4></p>
 
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<p>
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</br><p><h4>06/10/2014</h4></p>
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</p>
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 +
 
<p>Codes for IDT known. MEGAGEM2014 - 25% off one order, up to $800
<p>Codes for IDT known. MEGAGEM2014 - 25% off one order, up to $800
</p>
</p>
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<p>
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</p>
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<p>GBlocks designed to be compatible with biobricks and GoldenBraid (GB).
<p>GBlocks designed to be compatible with biobricks and GoldenBraid (GB).
</p>
</p>
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<p>
 
-
</p>
 
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<p><h4>06/11/2014</h4></p>
 
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<p>
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</br><p><h4>06/11/2014</h4></p>
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</p>
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 +
 
<p>We ordered the following gBlocks and primers.
<p>We ordered the following gBlocks and primers.
</p>
</p>
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<p>
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</p>
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<ul><li>EaDAcT: Eunymus alatus (adapted for GB) 1127 bp
<ul><li>EaDAcT: Eunymus alatus (adapted for GB) 1127 bp
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</table>
</table>
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<p>
 
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</p>
 
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<p><h4>06/24/2014</h4></p>
 
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<p>
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</br><p><h4>06/24/2014</h4></p>
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</p>
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 +
 
<p>We thought which parts of the GB collection could we use.
<p>We thought which parts of the GB collection could we use.
</p>
</p>
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<p>
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</p>
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<p>Strategy 1:
<p>Strategy 1:
</p>
</p>
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<p>
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</p>
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<ul><li>pP35S, pT35s (x2)
<ul><li>pP35S, pT35s (x2)
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</p>
</p>
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<p>
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</p>
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<ul><li>pP35S, pT35s
<ul><li>pP35S, pT35s
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</p>
</p>
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<p>
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</p>
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<ul><li>pP35S, pT35s
<ul><li>pP35S, pT35s
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</p>
</p>
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<p>
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</p>
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<table style="width:300px">
<table style="width:300px">
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</table>
</table>
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<p>
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</p>
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<p>Later we will need:
<p>Later we will need:
</p>
</p>
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<p>
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</p>
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<table style="width:300px">
<table style="width:300px">
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</table>
</table>
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<p>
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</p>
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<p>Prepare plaques with antibiotics Kan, Spm, Amp
<p>Prepare plaques with antibiotics Kan, Spm, Amp
</p>
</p>
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<p>
 
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</p>
 
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<p>
 
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</p>
 
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<p><h4>06/25/2014</h4></p>
 
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<p>
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</p>
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</br><p><h4>06/25/2014</h4></p>
 +
 
 +
 
<p>Grow the selected pieces from the GB collection in liquid medium (performed in laminar air flow cabinet).
<p>Grow the selected pieces from the GB collection in liquid medium (performed in laminar air flow cabinet).
</p>
</p>
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<p>
 
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</p>
 
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<p>
 
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</p>
 
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<p><h4>06/26/2014</h4></p>
 
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<p>
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</p>
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</br><p><h4>06/26/2014</h4></p>
 +
 
 +
 
<p>Culture in agar Petri dish. 2 plaques: Amp and Kan.
<p>Culture in agar Petri dish. 2 plaques: Amp and Kan.
</p>
</p>
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<p>
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</p>
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<p>Minipreps with EZNA Plasmid DNA MiniKit I.
<p>Minipreps with EZNA Plasmid DNA MiniKit I.
</p>
</p>
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<p>
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-
</p>
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<p>Expected digestions:
<p>Expected digestions:
</p>
</p>
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<p>
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</p>
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<table style="width:300px">
<table style="width:300px">
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</table>
</table>
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<p>
 
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</p>
 
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<p>
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</p>
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<p>Electrophoresis analysis.
<p>Electrophoresis analysis.
</p>
</p>
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<p>
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</p>
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<img src=https://static.igem.org/mediawiki/2014/d/d9/20140626_piezas_coleccion.png
<img src=https://static.igem.org/mediawiki/2014/d/d9/20140626_piezas_coleccion.png
>
>
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<p>
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</p>
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<p>We got the expected bands in all cases.
<p>We got the expected bands in all cases.
</p>
</p>
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<p>
 
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</p>
 
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<p>
 
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</p>
 
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<p><h4>01/07/2014</h4></p>
 
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<p>
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</p>
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</br><p><h4>01/07/2014</h4></p>
 +
 
 +
 
<p>AtrD11 amplification by PCR with primers that contain extra nucleotides to introduce them in the sequence.  
<p>AtrD11 amplification by PCR with primers that contain extra nucleotides to introduce them in the sequence.  
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</p>
</p>
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<p>
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</p>
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<p>Reagents needed:
<p>Reagents needed:
</p>
</p>
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<p>
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</p>
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<ul><li>32.5 uL of H2O miliQ
<ul><li>32.5 uL of H2O miliQ
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</p>
</p>
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<p>
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</p>
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<p>Electrophoresis performed to check the PCR product, which was expected to be around 1 kb.  
<p>Electrophoresis performed to check the PCR product, which was expected to be around 1 kb.  
</p>
</p>
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<p>
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</p>
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<img src=https://static.igem.org/mediawiki/2014/6/6a/20140701_pcr_gblock_atrd11.png
<img src=https://static.igem.org/mediawiki/2014/6/6a/20140701_pcr_gblock_atrd11.png
>
>
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<p>
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</p>
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<p>pUPD ligation of EaDAct, HarFar and Atr∆11
<p>pUPD ligation of EaDAct, HarFar and Atr∆11
</p>
</p>
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<p>
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</p>
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<p>Reagents needed for the reaction of ligation:
<p>Reagents needed for the reaction of ligation:
</p>
</p>
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<p>
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</p>
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<ul><li>1 uL pUPD
<ul><li>1 uL pUPD
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</p>
</p>
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<p>
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</p>
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<p>Termocycler parameters: The ligase temperature was 16º C and the BsmBI temperature was 37º C.  
<p>Termocycler parameters: The ligase temperature was 16º C and the BsmBI temperature was 37º C.  
</p>
</p>
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<p>
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</p>
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<p>As a result, there are obtained three different pUPD plasmids containing the genes EaDacT, HarFAR and Atr∆11.
<p>As a result, there are obtained three different pUPD plasmids containing the genes EaDacT, HarFAR and Atr∆11.
</p>
</p>
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<p>
 
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</p>
 
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<p>
 
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</p>
 
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<p><h4>07/02/2014</h4></p>
 
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<p>
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</p>
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</br><p><h4>07/02/2014</h4></p>
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 +
 
<p>E. coli transformation
<p>E. coli transformation
</p>
</p>
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<p>
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</p>
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<p>This step is performed in a laminar air flow cabinet (LAF).  
<p>This step is performed in a laminar air flow cabinet (LAF).  
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</p>
</p>
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<p>
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</p>
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<p>Reagents needed:
<p>Reagents needed:
</p>
</p>
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<p>
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</p>
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<ul><li>E. coli aliquot
<ul><li>E. coli aliquot
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</p>
</p>
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<p>
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</p>
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<p>After incubation, culture in Petri plates (always in a LAF).
<p>After incubation, culture in Petri plates (always in a LAF).
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</p>
</p>
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<p>
 
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</p>
 
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<p><h4>07/03/2014</h4></p>
 
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<p>
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</br><p><h4>07/03/2014</h4></p>
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</p>
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 +
 
<p>Transformed colonies selection. The white ones are recultured in liquid medium. One colony of each transformation is picked and cultured in 3.5 mL LB and 7 uL Amp. This step is repeated three times:
<p>Transformed colonies selection. The white ones are recultured in liquid medium. One colony of each transformation is picked and cultured in 3.5 mL LB and 7 uL Amp. This step is repeated three times:
</p>
</p>
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<p>
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</p>
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<ul><li>3x 1 colony of EaDacT in pUPD + LB + Amp
<ul><li>3x 1 colony of EaDacT in pUPD + LB + Amp
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</p>
</p>
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<p>
 
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</p>
 
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<p>
 
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</p>
 
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<p><h4>07/04/2014</h4></p>
 
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<p>
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</p>
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</br><p><h4>07/04/2014</h4></p>
 +
 
 +
 
<p>Digestions in silico using Vector NTI to check after minipreps if ligations are correct.
<p>Digestions in silico using Vector NTI to check after minipreps if ligations are correct.
</p>
</p>
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<p>
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</p>
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<table style="width:300px">
<table style="width:300px">
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</table>
</table>
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<p>
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</p>
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<p>Digestion reagents:
<p>Digestion reagents:
</p>
</p>
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<p>
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</p>
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<ul><li>0.5 uL restriction enzyme
<ul><li>0.5 uL restriction enzyme
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</p>
</p>
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<p>
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</p>
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<ul><li>Master mix for NotI
<ul><li>Master mix for NotI
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</p>
</p>
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<p>
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</p>
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<img src=https://static.igem.org/mediawiki/2014/d/d5/20140704_digestiones_ligaciones.png
<img src=https://static.igem.org/mediawiki/2014/d/d5/20140704_digestiones_ligaciones.png
>
>
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<p>
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</p>
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<p>Comments:
<p>Comments:
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</ul>
</ul>
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<p><h4>07/06/2014</h4></p>
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</br><p><h4>07/06/2014</h4></p>
 +
 
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<p>
 
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</p>
 
<p>We picked again 3 colonies for each construction, and we made sure that we picked the WHITE ones. We cultivated them in a "double check" (name invented by us)  liquid medium. Those tubes contain:
<p>We picked again 3 colonies for each construction, and we made sure that we picked the WHITE ones. We cultivated them in a "double check" (name invented by us)  liquid medium. Those tubes contain:
</p>
</p>
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<p>
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</p>
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<ul><li>3.5 mL LB
<ul><li>3.5 mL LB
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</ul>
</ul>
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<p><h4>07/07/2014</h4></p>
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</br><p><h4>07/07/2014</h4></p>
 +
 
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<p>
 
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</p>
 
<p>We made minipreps of yesterday's culture. Thanks to our "double check" medium we found which colonies were well picked. Finally we had minipreps of tubes HarFAR 1, 2, 3; EaDAcT 3 and AtrD11 2, 3.
<p>We made minipreps of yesterday's culture. Thanks to our "double check" medium we found which colonies were well picked. Finally we had minipreps of tubes HarFAR 1, 2, 3; EaDAcT 3 and AtrD11 2, 3.
</p>
</p>
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<p>
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</p>
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<p>Once we had the minipreps, we perform the digestions to check which were correct and send them to sequencing. This time we selected RsaI instead of BanII. The in silico digestions were as follows.
<p>Once we had the minipreps, we perform the digestions to check which were correct and send them to sequencing. This time we selected RsaI instead of BanII. The in silico digestions were as follows.
</p>
</p>
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<p>
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</p>
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<table style="width:300px">
<table style="width:300px">
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</table>
</table>
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<p>
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</p>
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<p>Preparation of master mixes
<p>Preparation of master mixes
</p>
</p>
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<p>
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</p>
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<ul><li>Master mix for NotI
<ul><li>Master mix for NotI
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</p>
</p>
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<p>
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</p>
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<img src=https://static.igem.org/mediawiki/2014/c/ca/20140707_digestiones_ligaciones2.png
<img src=https://static.igem.org/mediawiki/2014/c/ca/20140707_digestiones_ligaciones2.png
>
>
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<p>
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</p>
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<p>Everything was OK. We sent AtrD11 (3), HarFAR (3) and EaDAcT (3) to sequence.
<p>Everything was OK. We sent AtrD11 (3), HarFAR (3) and EaDAcT (3) to sequence.
</p>
</p>
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<p>
 
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</p>
 
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<p><h4>07/08/2014</h4></p>
 
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<p>
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</br><p><h4>07/08/2014</h4></p>
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</p>
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 +
 
<p>Now, while we wait for sequencing results, we go on as they were going to be correct in order to save time.
<p>Now, while we wait for sequencing results, we go on as they were going to be correct in order to save time.
</p>
</p>
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<p>
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</p>
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<p>The next step is to build a transciptional unit (TU) with our sequences. A transcriptional unit is a structure composed by promoter, coding sequence (CDS) and terminator in an alpha or omega vector.
<p>The next step is to build a transciptional unit (TU) with our sequences. A transcriptional unit is a structure composed by promoter, coding sequence (CDS) and terminator in an alpha or omega vector.
</p>
</p>
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<p>
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</p>
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<p>Reagents needed for ligation:
<p>Reagents needed for ligation:
</p>
</p>
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<p>
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</p>
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<ul><li>1 uL promoter 75 ng/uL
<ul><li>1 uL promoter 75 ng/uL
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</p>
</p>
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<p>
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</p>
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<p>Take into account that if we want to make binary constructions later (merge 2 TU in a same vector), we need to clone each TU in a different alpha vector.
<p>Take into account that if we want to make binary constructions later (merge 2 TU in a same vector), we need to clone each TU in a different alpha vector.
</p>
</p>
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<p>
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</p>
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<p>Strategy Promoter-Terminator:
<p>Strategy Promoter-Terminator:
</p>
</p>
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<p>
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</p>
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<table style="width:300px">
<table style="width:300px">
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</table>
</table>
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<p>
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</p>
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<p>Adjust concentrations to 75 ng/uL for ligation reaction
<p>Adjust concentrations to 75 ng/uL for ligation reaction
</p>
</p>
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<p>
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</p>
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<p>Initial concentrations (nanodrop):
<p>Initial concentrations (nanodrop):
</p>
</p>
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<p>
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</p>
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<table style="width:300px">
<table style="width:300px">
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</table>
</table>
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<p>
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</p>
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<p>Ligation reaction
<p>Ligation reaction
</p>
</p>
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<p>
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</p>
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<ul><li>P35s:AtrD11:T35s in 2alpha1
<ul><li>P35s:AtrD11:T35s in 2alpha1
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</ul></ul>
</ul></ul>
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<p><h4>07/09/2014</h4></p>
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</br><p><h4>07/09/2014</h4></p>
 +
 
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<p>
 
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</p>
 
<p>Transformation of constructions in E. coli
<p>Transformation of constructions in E. coli
</p>
</p>
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<p>
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</p>
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<p>We finally got the sequencing results from 07/07/2014.
<p>We finally got the sequencing results from 07/07/2014.
</p>
</p>
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<p>
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</p>
+
<ul><li>Mutation in AtrD11 -> We threw away the colonies and transformed cells. We picked again white colonies.
<ul><li>Mutation in AtrD11 -> We threw away the colonies and transformed cells. We picked again white colonies.
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</p>
</p>
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<p>
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</p>
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<ul><li>Worked in the LAF
<ul><li>Worked in the LAF
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</p>
</p>
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<p>
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</p>
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<ul><li>50 mL transformation in one plaque
<ul><li>50 mL transformation in one plaque
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</ul>
</ul>
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<p><h4>07/10/2014</h4></p>
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</br><p><h4>07/10/2014</h4></p>
 +
 
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<p>
 
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</p>
 
<p>Pick colonies and grow them in liquid medium.
<p>Pick colonies and grow them in liquid medium.
</p>
</p>
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<p>
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</p>
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<ul><li>6 from AtrD11 (repetition because of mutation)
<ul><li>6 from AtrD11 (repetition because of mutation)
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</p>
</p>
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<p>
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</p>
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<p>We have checked the promoters and terminators are both compatible with GB and BioBricks.
<p>We have checked the promoters and terminators are both compatible with GB and BioBricks.
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</p>
</p>
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<p>
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</p>
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<p>Pick 3 colonies of P35s:HarFAR:THsp and PAtUbq:EaDAcT:THsp. Culture in liquid medium with Kan.
<p>Pick 3 colonies of P35s:HarFAR:THsp and PAtUbq:EaDAcT:THsp. Culture in liquid medium with Kan.
</p>
</p>
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<p>
 
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</p>
 
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<p><h4>07/11/2014</h4></p>
 
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<p>
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</br><p><h4>07/11/2014</h4></p>
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</p>
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<p>We made minipreps of yesterday's liquid culture. Thanks to our "double check" medium we found which colonies were well picked. Finally we had minipreps of tubes AtrD11 3 and 6; 2omega1; 2omega2; constructions P35s:HarFAR:TatHSP 1, 2, 3 and PAtUbq:EaDAcT:TatHSP 1, 2, 3.
<p>We made minipreps of yesterday's liquid culture. Thanks to our "double check" medium we found which colonies were well picked. Finally we had minipreps of tubes AtrD11 3 and 6; 2omega1; 2omega2; constructions P35s:HarFAR:TatHSP 1, 2, 3 and PAtUbq:EaDAcT:TatHSP 1, 2, 3.
</p>
</p>
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<p>
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</p>
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<p>Additionally, we have cultured each of the colonies named above to store them.
<p>Additionally, we have cultured each of the colonies named above to store them.
</p>
</p>
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<p>
 
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</p>
 
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<p><h4>07/14/2014</h4></p>
 
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<p>
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</br><p><h4>07/14/2014</h4></p>
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</p>
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 +
 
<p>We tested the minipreps made last friday by digestion. Once they were checked, we send the correct ones to sequencing. The in silico digestions were as follows.
<p>We tested the minipreps made last friday by digestion. Once they were checked, we send the correct ones to sequencing. The in silico digestions were as follows.
</p>
</p>
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<p>
+
 
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</p>
+
<table style="width:300px">
<table style="width:300px">
Line 1,205: Line 1,106:
</table>
</table>
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<p>
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</p>
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<p>Preparation of master mixes
<p>Preparation of master mixes
</p>
</p>
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<p>
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</p>
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<ul><li>Master mix for HindIII
<ul><li>Master mix for HindIII
Line 1,264: Line 1,163:
</ul></ul>
</ul></ul>
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<p>
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</p>
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<p>We run the electrophoresis gel to check if this time we have done it correctly.
<p>We run the electrophoresis gel to check if this time we have done it correctly.
</p>
</p>
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<p>
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</p>
+
<img src=https://static.igem.org/mediawiki/2014/7/7a/20140714_digestion_ligaciones.png
<img src=https://static.igem.org/mediawiki/2014/7/7a/20140714_digestion_ligaciones.png
>
>
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<p>
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</p>
+
<p>Everything was OK except the AtrD11 (3), which had some partial digestion. It was the reason we sent AtrD11 (6) to sequence.
<p>Everything was OK except the AtrD11 (3), which had some partial digestion. It was the reason we sent AtrD11 (6) to sequence.
</p>
</p>
-
<p>
 
-
</p>
 
-
<p><h4>07/15/2014</h4></p>
 
-
<p>
+
</br><p><h4>07/15/2014</h4></p>
-
</p>
+
 
 +
 
<p>We got the sequencing results from yesterday and everything was OK, so we made the transcriptional units ligation.  
<p>We got the sequencing results from yesterday and everything was OK, so we made the transcriptional units ligation.  
</p>
</p>
-
<p>
+
 
-
</p>
+
<p>Reagents needed for the reaction of ligation (Total volume = 12 uL):
<p>Reagents needed for the reaction of ligation (Total volume = 12 uL):
</p>
</p>
-
<p>
+
 
-
</p>
+
<ul><li>P35s:AtrD11:T35s in 2alpha1
<ul><li>P35s:AtrD11:T35s in 2alpha1
Line 1,388: Line 1,280:
</p>
</p>
-
<p>
+
 
-
</p>
+
<p>Finally, we prepared liquid cultures in order to store in glicerol. The tubes we used and their respective antibiotics were:
<p>Finally, we prepared liquid cultures in order to store in glicerol. The tubes we used and their respective antibiotics were:
</p>
</p>
-
<p>
+
 
-
</p>
+
<ul><li>Amp
<ul><li>Amp
Line 1,420: Line 1,310:
</ul></ul>
</ul></ul>
-
<p><h4>07/16/2014</h4></p>
+
</br><p><h4>07/16/2014</h4></p>
 +
 
-
<p>
 
-
</p>
 
<p>Storage in glycerol of the HarFAR (GB1018), AtrD11 (GB1019), EaDAcT (GB1020), P35s:HarFAR:TatHSP in 2alpha2 (GB1021) and PAtUbq:EaDAcT:TatHSP in 2alpha2 (GB1022) . we made 3 tubes: one for us, one for the GB collenction and one for reserve.  
<p>Storage in glycerol of the HarFAR (GB1018), AtrD11 (GB1019), EaDAcT (GB1020), P35s:HarFAR:TatHSP in 2alpha2 (GB1021) and PAtUbq:EaDAcT:TatHSP in 2alpha2 (GB1022) . we made 3 tubes: one for us, one for the GB collenction and one for reserve.  
</p>
</p>
-
<p>
+
 
-
</p>
+
<p>The procedure is to mix 700 uL of culture with 300 uL of glycerol 50%, spin it and store it in the -80ºC.
<p>The procedure is to mix 700 uL of culture with 300 uL of glycerol 50%, spin it and store it in the -80ºC.
</p>
</p>
-
<p>
 
-
</p>
 
-
<p><h4>07/17/2014</h4></p>
 
-
<p>
+
</br><p><h4>07/17/2014</h4></p>
-
</p>
+
 
 +
 
<p>Pick 3 colonies of P35s:AtrD11:T35s, P35s:HarFAR:T35s and P35s:EaDAcT:T35s. Culture in liquid medium with Kan.
<p>Pick 3 colonies of P35s:AtrD11:T35s, P35s:HarFAR:T35s and P35s:EaDAcT:T35s. Culture in liquid medium with Kan.
</p>
</p>
-
<p>
+
 
-
</p>
+
<p>Digestions in silico.
<p>Digestions in silico.
</p>
</p>
-
<p>
+
 
-
</p>
+
<table style="width:300px">
<table style="width:300px">
Line 1,479: Line 1,363:
</table>
</table>
-
<p>
+
 
-
</p>
+
<p>Preparation of reagents needed for genomic extraction of Candida tropicalis for FAO1.
<p>Preparation of reagents needed for genomic extraction of Candida tropicalis for FAO1.
</p>
</p>
-
<p>
 
-
</p>
 
-
<p><h4>07/18/2014</h4></p>
 
-
<p>
+
</br><p><h4>07/18/2014</h4></p>
-
</p>
+
 
 +
 
<p>Mistake in P35s:AtrD11:T35s, P35s:HarFAR:T35s and P35s:EaDAcT:T35s minipreps. Repeat tomorrow.
<p>Mistake in P35s:AtrD11:T35s, P35s:HarFAR:T35s and P35s:EaDAcT:T35s minipreps. Repeat tomorrow.
</p>
</p>
-
<p>
 
-
</p>
 
-
<p>
 
-
</p>
 
-
<p><h4>07/19/2014</h4></p>
 
-
<p>
+
 
-
</p>
+
</br><p><h4>07/19/2014</h4></p>
 +
 
 +
 
<p>Minipreps of P35s:AtrD11:T35s, P35s:HarFAR:T35s and P35s:EaDAcT:T35s. Concentration measuments with nanodrop.
<p>Minipreps of P35s:AtrD11:T35s, P35s:HarFAR:T35s and P35s:EaDAcT:T35s. Concentration measuments with nanodrop.
</p>
</p>
-
<p>
+
 
-
</p>
+
<table style="width:300px">
<table style="width:300px">
Line 1,556: Line 1,433:
</table>
</table>
-
<p>
+
 
-
</p>
+
<p>Digestions of P35s:AtrD11:T35s, P35s:HarFAR:T35s and P35s:EaDAcT:T35s and gel electrophoresis to check if transciptional units have been assembled OK.
<p>Digestions of P35s:AtrD11:T35s, P35s:HarFAR:T35s and P35s:EaDAcT:T35s and gel electrophoresis to check if transciptional units have been assembled OK.
</p>
</p>
-
<p>
+
 
-
</p>
+
<img src=https://static.igem.org/mediawiki/2014/3/3c/20140719_digestiones_TU.png
<img src=https://static.igem.org/mediawiki/2014/3/3c/20140719_digestiones_TU.png
>
>
-
<p>
+
 
-
</p>
+
<p>All digestions were correct except P35s:EaDAcT:T35s (2).
<p>All digestions were correct except P35s:EaDAcT:T35s (2).
</p>
</p>
-
<p>
+
 
-
</p>
+
<p>Ligation in omega vectors.
<p>Ligation in omega vectors.
</p>
</p>
-
<p>
+
 
-
</p>
+
<ul><li>P35s:AtrD11:T35s + P35s:HarFAR:T35s in 2omega1
<ul><li>P35s:AtrD11:T35s + P35s:HarFAR:T35s in 2omega1
Line 1,636: Line 1,508:
</p>
</p>
-
<p>
+
 
-
</p>
+
<p>Omega vectors include a resistance to spectinomycin.
<p>Omega vectors include a resistance to spectinomycin.
</p>
</p>
-
<p>
 
-
</p>
 
-
<p><h4>07/20/2014</h4></p>
 
-
<p>
+
</br><p><h4>07/20/2014</h4></p>
-
</p>
+
 
 +
 
<p>Transform and grow in Petri dishes yesterday's ligations: P35S:AtrD11:T35S + P35S:HarFAR:T35S in 2omega1 and P35S:EaDAcT:T35S in 2omega2.
<p>Transform and grow in Petri dishes yesterday's ligations: P35S:AtrD11:T35S + P35S:HarFAR:T35S in 2omega1 and P35S:EaDAcT:T35S in 2omega2.
</p>
</p>
-
<p>
 
-
</p>
 
-
<p><h4>07/21/2014</h4></p>
 
-
<p>
+
</br><p><h4>07/21/2014</h4></p>
-
</p>
+
 
 +
 
<p>Pick colonies of P35S:AtrD11:T35S + P35S:HarFAR:T35S in 2omega1 (3) and P35S:EaDAcT:T35S in 2omega2 (2).
<p>Pick colonies of P35S:AtrD11:T35S + P35S:HarFAR:T35S in 2omega1 (3) and P35S:EaDAcT:T35S in 2omega2 (2).
</p>
</p>
-
<p>
 
-
</p>
 
-
<p><h4>07/22/2014</h4></p>
 
-
<p>
+
</br><p><h4>07/22/2014</h4></p>
-
</p>
+
 
 +
 
<p>We made minipreps of yesterday's liquid culture. Selected tubes:  
<p>We made minipreps of yesterday's liquid culture. Selected tubes:  
Line 1,686: Line 1,551:
</p>
</p>
-
<p>
+
 
-
</p>
+
<p>Digestions in silico made to check the transcriptional units:
<p>Digestions in silico made to check the transcriptional units:
</p>
</p>
-
<p>
+
 
-
</p>
+
<table style="width:300px">
<table style="width:300px">
Line 1,714: Line 1,577:
</table>
</table>
-
<p>
+
 
-
</p>
+
<p>Digestion master mixes:
<p>Digestion master mixes:
</p>
</p>
-
<p>
+
 
-
</p>
+
<ul><li>Master mix for NotI
<ul><li>Master mix for NotI
Line 1,776: Line 1,637:
</p>
</p>
-
<p>
+
 
-
</p>
+
<p>All digestions were correct except the transcriptional unit of EaDAct in 2omega2 (P35s:EaDAcT:T35S).  
<p>All digestions were correct except the transcriptional unit of EaDAct in 2omega2 (P35s:EaDAcT:T35S).  
</p>
</p>
-
<p>
 
-
</p>
 
-
<p>
+
 
-
</p>
+
 
<p>*Aquí falta la imagen de las digestiones 22/07
<p>*Aquí falta la imagen de las digestiones 22/07
</p>
</p>
-
<p>
 
-
</p>
 
-
<p>
 
-
</p>
 
-
<p>
 
-
</p>
 
-
<p>
+
 
-
</p>
+
 
 +
 
 +
 
<p>Miniprep quantification:
<p>Miniprep quantification:
</p>
</p>
-
<p>
+
 
-
</p>
+
<table style="width:300px">
<table style="width:300px">
Line 1,837: Line 1,690:
</table>
</table>
-
<p>
+
 
-
</p>
+
<p>All of the pieces named above were adjusted at 75 ng/uL.
<p>All of the pieces named above were adjusted at 75 ng/uL.
</p>
</p>
-
<p>
+
 
-
</p>
+
<table style="width:300px">
<table style="width:300px">
Line 1,874: Line 1,725:
</table>
</table>
-
<p>
+
 
-
</p>
+
<p>As the digestions of the transcriptional unit (TU) of EaDAcT were incorrect, we repeated the process from the ligation step.  
<p>As the digestions of the transcriptional unit (TU) of EaDAcT were incorrect, we repeated the process from the ligation step.  
</p>
</p>
-
<p>
+
 
-
</p>
+
<p>We transformed the same TU in a E. coli competent strain (DH5alpha). Then, the transformants were cultured in LB media and Spm and stored at 37 ºC overnight.  
<p>We transformed the same TU in a E. coli competent strain (DH5alpha). Then, the transformants were cultured in LB media and Spm and stored at 37 ºC overnight.  
</p>
</p>
-
<p>
+
 
-
</p>
+
<p>Pieces taken from the GoldenBraid 2.0 collection were cultured in solid growth media:
<p>Pieces taken from the GoldenBraid 2.0 collection were cultured in solid growth media:
</p>
</p>
-
<p>
+
 
-
</p>
+
<p>With Amp:
<p>With Amp:
Line 1,923: Line 1,770:
</p>
</p>
-
<p>
 
-
</p>
 
-
<p><h4>07/23/2014</h4></p>
 
-
<p>
+
</br><p><h4>07/23/2014</h4></p>
-
</p>
+
 
 +
 
<p>Candida genome extraction
<p>Candida genome extraction
</p>
</p>
-
<p>
+
 
-
</p>
+
<p>Cultures measured at 600 nm:
<p>Cultures measured at 600 nm:
Line 1,951: Line 1,795:
</p>
</p>
-
<p>
+
 
-
</p>
+
<p>The absorbance was measured again:
<p>The absorbance was measured again:
Line 1,968: Line 1,811:
</p>
</p>
-
<p>
+
 
-
</p>
+
<ul><li>Grow yeast in 2 or 5 mL YPD to exponential phase.  
<ul><li>Grow yeast in 2 or 5 mL YPD to exponential phase.  
Line 2,018: Line 1,860:
</p>
</p>
-
<p>
+
 
-
</p>
+
<table style="width:300px">
<table style="width:300px">
Line 2,034: Line 1,875:
</table>
</table>
-
<p>
+
 
-
</p>
+
<p>Electrophoresis made for check the extraction quality was not correct.  
<p>Electrophoresis made for check the extraction quality was not correct.  
</p>
</p>
-
<p>
+
 
-
</p>
+
<p>* Añadir imagen electroforesis extracción genomas candida y saccharomyces (23/07)
<p>* Añadir imagen electroforesis extracción genomas candida y saccharomyces (23/07)
</p>
</p>
-
<p>
 
-
</p>
 
-
<p>
+
 
-
</p>
+
 
<p>EaDAcT and collection pieces made yesterday were recultured in liquid media.
<p>EaDAcT and collection pieces made yesterday were recultured in liquid media.
</p>
</p>
-
<p>
 
-
</p>
 
-
<p><h4>07/24/2014</h4></p>
 
-
<p>
+
</br><p><h4>07/24/2014</h4></p>
-
</p>
+
 
 +
 
<p>The genomic quality of both organisms (C. tropicalis and S. cerevisiae) was checked in an agarose gel:
<p>The genomic quality of both organisms (C. tropicalis and S. cerevisiae) was checked in an agarose gel:
</p>
</p>
-
<p>
+
 
-
</p>
+
<p>*Atención: incluir aquí el gel de geómico de candida y cerevisiae del 24/07
<p>*Atención: incluir aquí el gel de geómico de candida y cerevisiae del 24/07
</p>
</p>
-
<p>
+
 
-
</p>
+
<p>We got the Candida genome band, however, the Saccharomyces genome band was not present.
<p>We got the Candida genome band, however, the Saccharomyces genome band was not present.
</p>
</p>
-
<p>
 
-
</p>
 
-
<p>
+
 
-
</p>
+
 
<p>Additionally, minipreps of the liquid culture made yesterday were made and also recultured in solid agar plate.  
<p>Additionally, minipreps of the liquid culture made yesterday were made and also recultured in solid agar plate.  
</p>
</p>
-
<p>
+
 
-
</p>
+
<ul><li>P35S:EaDAcT:T35S in 2omega2 (tubes 1, 2, 3)
<ul><li>P35S:EaDAcT:T35S in 2omega2 (tubes 1, 2, 3)
Line 2,116: Line 1,946:
</p>
</p>
-
<p>
 
-
</p>
 
-
<p><h4>07/25/2014</h4></p>
 
-
<p>
+
</br><p><h4>07/25/2014</h4></p>
-
</p>
+
 
 +
 
<p>Digestions in silico made for checking yesterday's minipreps:
<p>Digestions in silico made for checking yesterday's minipreps:
</p>
</p>
-
<p>
+
 
-
</p>
+
<table style="width:300px">
<table style="width:300px">
Line 2,161: Line 1,988:
</table>
</table>
-
<p>
+
 
-
</p>
+
<p>Digestion master mixes:
<p>Digestion master mixes:
</p>
</p>
-
<p>
+
 
-
</p>
+
<ul><li>Master mix for NotI
<ul><li>Master mix for NotI
Line 2,223: Line 2,048:
</p>
</p>
-
<p>
+
 
-
</p>
+
<p>*¡Atención! Aquí falta un gel (grande) con el resulado de las digestiones de las piezas de la colección y de la Ea
<p>*¡Atención! Aquí falta un gel (grande) con el resulado de las digestiones de las piezas de la colección y de la Ea
</p>
</p>
-
<p>
+
 
-
</p>
+
<p>All pieces were correct except the TU corresponding to P35:EaDAcT:T35S.
<p>All pieces were correct except the TU corresponding to P35:EaDAcT:T35S.
</p>
</p>
-
<p>
 
-
</p>
 
-
<p><h4>07/28/2014</h4></p>
 
-
<p>
+
</br><p><h4>07/28/2014</h4></p>
-
</p>
+
 
 +
 
<p>Once the Candida tropicalis genome DNA is obtained, the FAO1 gene can be amplified by PCR.
<p>Once the Candida tropicalis genome DNA is obtained, the FAO1 gene can be amplified by PCR.
</p>
</p>
-
<p>
+
 
-
</p>
+
<p>PCR reaction reagents:
<p>PCR reaction reagents:
</p>
</p>
-
<p>
+
 
-
</p>
+
<table style="width:300px">
<table style="width:300px">
Line 2,304: Line 2,123:
</table>
</table>
-
<p>
+
 
-
</p>
+
<p>Annealing temperatures
<p>Annealing temperatures
Line 2,321: Line 2,139:
</p>
</p>
-
<p>
+
 
-
</p>
+
<ul><li>FAO1-PCR1: 1157 bp
<ul><li>FAO1-PCR1: 1157 bp
Line 2,335: Line 2,152:
>
>
-
<p>
+
 
-
</p>
+
<p>Both FAO1 PCR products were not correct.
<p>Both FAO1 PCR products were not correct.
</p>
</p>
-
<p>
+
 
-
</p>
+
<p>As the EaDAcT TU was not correct, ligation reaction was done again. The following table shows ligation details:
<p>As the EaDAcT TU was not correct, ligation reaction was done again. The following table shows ligation details:
</p>
</p>
-
<p>
+
 
-
</p>
+
<table style="width:300px">
<table style="width:300px">
Line 2,384: Line 2,198:
</table>
</table>
-
<p>
 
-
</p>
 
-
<p><h4>07/29/2014</h4></p>
 
-
<p>
+
</br><p><h4>07/29/2014</h4></p>
-
</p>
+
 
 +
 
<p>As the FAO1 PCR was not correct, we repeated the reaction. Below is a table showing the details:
<p>As the FAO1 PCR was not correct, we repeated the reaction. Below is a table showing the details:
</p>
</p>
-
<p>
+
 
-
</p>
+
<table style="width:300px">
<table style="width:300px">
Line 2,426: Line 2,237:
</table>
</table>
-
<p>
+
 
-
</p>
+
<p>PCR temperatures, 25 cycles:
<p>PCR temperatures, 25 cycles:
</p>
</p>
-
<p>
+
 
-
</p>
+
<table style="width:300px">
<table style="width:300px">
Line 2,457: Line 2,266:
</table>
</table>
-
<p>
+
 
-
</p>
+
<p>We made a mistake preparing the FAO1-PCR1 adding the wrong template, so we do not expect the correct FAO11-PCR1 product.  
<p>We made a mistake preparing the FAO1-PCR1 adding the wrong template, so we do not expect the correct FAO11-PCR1 product.  
</p>
</p>
-
<p>
+
 
-
</p>
+
<p>EaDAcT Transcriptional Unit (TU) transformation
<p>EaDAcT Transcriptional Unit (TU) transformation
</p>
</p>
-
<p>
+
 
-
</p>
+
<p>Using an electrocompetent E. coli strain (DH5alpha) and 1.5 ul ligation (P35S:EaDAcT:T35S in 2omega2), the mix is electroporated at 1500 V. Then, 300 uL of SOC are added and stored at 37 ºC with agitation.  
<p>Using an electrocompetent E. coli strain (DH5alpha) and 1.5 ul ligation (P35S:EaDAcT:T35S in 2omega2), the mix is electroporated at 1500 V. Then, 300 uL of SOC are added and stored at 37 ºC with agitation.  
</p>
</p>
-
<p>
 
-
</p>
 
-
<p><h4>07/30/2014</h4></p>
 
-
<p>
+
</br><p><h4>07/30/2014</h4></p>
-
</p>
+
-
<p>
 
-
</p>
 
-
<p>
 
-
</p>
 
-
<p>
 
-
</p>
 
-
<h3><p></p>Switch</h3>
 
-
<p><h4>07/04/2014</h4></p>
 
-
<p>
+
 
-
</p>
+
 
 +
 
 +
</br></br><h3><p></p>Switch</h3>
 +
 
 +
</br><p><h4>07/04/2014</h4></p>
 +
 
 +
 
<p>Adquisition of S. cerevisiae genomic DNA. (5 uL, stored in the fridge)
<p>Adquisition of S. cerevisiae genomic DNA. (5 uL, stored in the fridge)
</p>
</p>
-
<p>
 
-
</p>
 
-
<p><h4>07/28/2014</h4></p>
 
-
<p>
+
</br><p><h4>07/28/2014</h4></p>
-
</p>
+
 
 +
 
<p>We had the genome of S. cerevisiae, needed to extract the target genes that are going to be used to build the switch. However we finally used our genome extraction (see Biosynthesis part, date 07/23/2014 for further details).
<p>We had the genome of S. cerevisiae, needed to extract the target genes that are going to be used to build the switch. However we finally used our genome extraction (see Biosynthesis part, date 07/23/2014 for further details).
Line 2,522: Line 2,320:
</p>
</p>
-
<p>
+
 
-
</p>
+
<table style="width:300px">
<table style="width:300px">
Line 2,553: Line 2,350:
</table>
</table>
-
<p>
+
 
-
</p>
+
<p>Annealing temperature: both 61 ºC
<p>Annealing temperature: both 61 ºC
</p>
</p>
-
<p>
+
 
-
</p>
+
<p>PCR products were checked using an electrophoresis. Expected bands:
<p>PCR products were checked using an electrophoresis. Expected bands:
</p>
</p>
-
<p>
+
 
-
</p>
+
<ul><li>CUP1-PCR1: 386 bp
<ul><li>CUP1-PCR1: 386 bp
Line 2,579: Line 2,373:
>
>
-
<p>
+
 
-
</p>
+
<p>Both CUP1 and CUP2 PCR products were correct.
<p>Both CUP1 and CUP2 PCR products were correct.
</p>
</p>
-
<p>
 
-
</p>
 
-
<p>
 
-
</p>
 
-
<p>
 
-
</p>
 
-
<p>
 
-
</p>
 
-
<p>
 
-
</p>
 
-
<h3><p></p>Trichome promoter</h3>
 
-
<p><h4>07/03/2014</h4></p>
 
-
<p>
+
 
-
</p>
+
 
 +
 
 +
</br></br><h3><p></p>Trichome promoter</h3>
 +
 
 +
</br><p><h4>07/03/2014</h4></p>
 +
 
 +
 
<p>Genomic DNA extraction from Nicotiana tabacum. We need the genome of this organism because we want to obtain the trichome promoter from the NtCPS2 gene.
<p>Genomic DNA extraction from Nicotiana tabacum. We need the genome of this organism because we want to obtain the trichome promoter from the NtCPS2 gene.
</p>
</p>
-
<p>
+
 
-
</p>
+
<ul><li>Obtain 100 mg of the tobacco leaves (5 disks made with a 1.5 mL vial). Made it twice.
<ul><li>Obtain 100 mg of the tobacco leaves (5 disks made with a 1.5 mL vial). Made it twice.
Line 2,687: Line 2,473:
</p>
</p>
-
<p>
+
 
-
</p>
+
<ul><li>Tabacco 1: 182 ng/uL (Thrown away)
<ul><li>Tabacco 1: 182 ng/uL (Thrown away)
Line 2,701: Line 2,486:
</p>
</p>
-
<p>
+
 
-
</p>
+
<img src=https://static.igem.org/mediawiki/2014/5/5e/20140703_extraccion_genomico_tobacco.png
<img src=https://static.igem.org/mediawiki/2014/5/5e/20140703_extraccion_genomico_tobacco.png
>
>
-
<p>
+
 
-
</p>
+
<p>It is correct.
<p>It is correct.
</p>
</p>
-
<p>
 
-
</p>
 
-
<p><h4>07/10/2014</h4></p>
 
-
<p>
+
</br><p><h4>07/10/2014</h4></p>
-
</p>
+
 
 +
 
<p>PCR of genomic extraction of tobacco in order to amplify the trichome promoter CPS2.
<p>PCR of genomic extraction of tobacco in order to amplify the trichome promoter CPS2.
</p>
</p>
-
<p>
+
 
-
</p>
+
<p>Ordered primers
<p>Ordered primers
</p>
</p>
-
<p>
+
 
-
</p>
+
<ul><li>IGEMJULO1
<ul><li>IGEMJULO1
Line 2,744: Line 2,523:
</p>
</p>
-
<p>
+
 
-
</p>
+
<ul><li>IGEMJUL01 + 566 uL miliQ H2O
<ul><li>IGEMJUL01 + 566 uL miliQ H2O
Line 2,758: Line 2,536:
</p>
</p>
-
<p>
+
 
-
</p>
+
<p>Reagents needed for PCR:
<p>Reagents needed for PCR:
</p>
</p>
-
<p>
+
 
-
</p>
+
<ul><li>0.5 uL template
<ul><li>0.5 uL template
Line 2,793: Line 2,569:
</p>
</p>
-
<p>
+
 
-
</p>
+
<p>Parameters:
<p>Parameters:
</p>
</p>
-
<p>
+
 
-
</p>
+
<ul><li>98 ºC (2 min)
<ul><li>98 ºC (2 min)
Line 2,825: Line 2,599:
>
>
-
<p>
+
 
-
</p>
+
<p>We didn't get PCR product.
<p>We didn't get PCR product.
</p>
</p>
-
<p>
 
-
</p>
 
-
<p><h4>07/11/2014</h4></p>
 
-
<p>
+
</br><p><h4>07/11/2014</h4></p>
-
</p>
+
 
 +
 
<p>Repeat PCR with different parameters.
<p>Repeat PCR with different parameters.
</p>
</p>
-
<p>
+
 
-
</p>
+
<p> 1 2 3 4 5
<p> 1 2 3 4 5
Line 2,869: Line 2,639:
</p>
</p>
-
<p>
+
 
-
</p>
+
<p>1, 2 and 5 contain buffer F; 3 and 4 contain buffer GC.
<p>1, 2 and 5 contain buffer F; 3 and 4 contain buffer GC.
</p>
</p>
-
<p>
+
 
-
</p>
+
<p>PCR parameters
<p>PCR parameters
</p>
</p>
-
<p>
+
 
-
</p>
+
<ul><li>98 ºC (2 min)
<ul><li>98 ºC (2 min)
Line 2,907: Line 2,674:
>
>
-
<p>
+
 
-
</p>
+
<p>No PCR products again.
<p>No PCR products again.
</p>
</p>
-
<p>
+
 
-
</p>
+
<p>Repeat PCR again with other parameters.
<p>Repeat PCR again with other parameters.
</p>
</p>
-
<p>
+
 
-
</p>
+
<p> Buffer HF Buffer GC
<p> Buffer HF Buffer GC
Line 2,946: Line 2,710:
</p>
</p>
-
<p>
+
 
-
</p>
+
<p>Set 4 tubes with each buffer at different temperatures: 49, 52, 55, 60.
<p>Set 4 tubes with each buffer at different temperatures: 49, 52, 55, 60.
</p>
</p>
-
<p>
+
 
-
</p>
+
<ul><li>98 ºC (2 min)
<ul><li>98 ºC (2 min)
Line 2,978: Line 2,740:
>
>
-
<p>
+
 
-
</p>
+
<p>No PCR products again.
<p>No PCR products again.
</p>
</p>
-
<p>
 
-
</p>
 
-
<p><h4>07/14/2014</h4></p>
 
-
<p>
+
</br><p><h4>07/14/2014</h4></p>
-
</p>
+
 
 +
 
<p>Repeat PCR again with more genomic.
<p>Repeat PCR again with more genomic.
</p>
</p>
-
<p>
+
 
-
</p>
+
<table style="width:300px">
<table style="width:300px">
Line 3,026: Line 2,784:
</table>
</table>
-
<p>
+
 
-
</p>
+
<p>Same parameters as before except annealing temperatures which are: 50, 53, 57, 59  ºC.
<p>Same parameters as before except annealing temperatures which are: 50, 53, 57, 59  ºC.
</p>
</p>
-
<p>
+
 
-
</p>
+
<img src=https://static.igem.org/mediawiki/2014/3/3a/20140714_productoPCR_tricomas_tercera_repeticion.png
<img src=https://static.igem.org/mediawiki/2014/3/3a/20140714_productoPCR_tricomas_tercera_repeticion.png
>
>
-
<p>
+
 
-
</p>
+
<p>We still without having any amplification.
<p>We still without having any amplification.
</p>
</p>
-
<p>
 
-
</p>
 
-
<p><h4>07/18/2014</h4></p>
 
-
<p>
+
</br><p><h4>07/18/2014</h4></p>
-
</p>
+
 
 +
 
<p>Repeat the PCR with other enzyme.
<p>Repeat the PCR with other enzyme.
</p>
</p>
-
<p>
+
 
-
</p>
+
<ul><li>12.5 uL Q5 Master mix (2x).
<ul><li>12.5 uL Q5 Master mix (2x).
Line 3,078: Line 2,830:
</p>
</p>
-
<p>
+
 
-
</p>
+
<ul><li>98 ºC (30 sec)
<ul><li>98 ºC (30 sec)
Line 3,104: Line 2,855:
>
>
-
<p>
+
 
-
</p>
+
<p>The DNA fragment of interest is around 1.5 kb so we see we finally obtained amplification at 55 and 59 ºC reactions.
<p>The DNA fragment of interest is around 1.5 kb so we see we finally obtained amplification at 55 and 59 ºC reactions.
</p>
</p>
-
<p>
 
-
</p>
 
-
<p><h4>07/19/2014</h4></p>
 
-
<p>
+
</br><p><h4>07/19/2014</h4></p>
-
</p>
+
 
 +
 
<p>Trichome promoter PCR product ligation in pUPD.
<p>Trichome promoter PCR product ligation in pUPD.
</p>
</p>
-
<p>
+
 
-
</p>
+
<ul><li>1 uL pUPD
<ul><li>1 uL pUPD
Line 3,147: Line 2,894:
</p>
</p>
-
<p>
 
-
</p>
 
-
<p><h4>07/20/2014</h4></p>
 
-
<p>
+
</br><p><h4>07/20/2014</h4></p>
-
</p>
+
 
 +
 
<p>Transform and grow in Petri dishes yesterday's ligation of the trichome promoter in pUPD.
<p>Transform and grow in Petri dishes yesterday's ligation of the trichome promoter in pUPD.
</p>
</p>
-
<p>
 
-
</p>
 
-
<p><h4>07/21/2014</h4></p>
 
-
<p>
+
</br><p><h4>07/21/2014</h4></p>
-
</p>
+
 
 +
 
<p>We picked colonies of the trichome promoter in pUPD and grown it in liquid culture.
<p>We picked colonies of the trichome promoter in pUPD and grown it in liquid culture.
</p>
</p>
-
<p>
 
-
</p>
 
-
<p><h4>07/22/2014</h4></p>
 
-
<p>
+
</br><p><h4>07/22/2014</h4></p>
-
</p>
+
 
 +
 
<p>We made minipreps of yesterday's liquid culture. Additionally, we have recultured them in solid growth media.  
<p>We made minipreps of yesterday's liquid culture. Additionally, we have recultured them in solid growth media.  
</p>
</p>
-
<p>
+
 
-
</p>
+
<p>Digestions in silico made to check the insertion:
<p>Digestions in silico made to check the insertion:
</p>
</p>
-
<p>
+
 
-
</p>
+
<table style="width:300px">
<table style="width:300px">
Line 3,202: Line 2,941:
</table>
</table>
-
<p>
+
 
-
</p>
+
<p>Note: To see further details of digestion master mixes, go to the biosynthesis part, date 07/22/2014.
<p>Note: To see further details of digestion master mixes, go to the biosynthesis part, date 07/22/2014.
</p>
</p>
-
<p>
 
-
</p>
 
-
<p><h4>07/23/2014</h4></p>
 
-
<p>
+
</br><p><h4>07/23/2014</h4></p>
-
</p>
+
 
 +
 
<p>Yesterday's digestions were correct, so the trichome promoter in pUPD was send to sequencing.
<p>Yesterday's digestions were correct, so the trichome promoter in pUPD was send to sequencing.
</p>
</p>
-
<p>
 
-
</p>
 
-
<p><h4>07/24/2014</h4></p>
 
-
<p>
+
</br><p><h4>07/24/2014</h4></p>
-
</p>
+
 
 +
 
<p>Results of sequencing the promoter were obtained:
<p>Results of sequencing the promoter were obtained:
</p>
</p>
-
<p>
+
 
-
</p>
+
<table style="width:300px">
<table style="width:300px">
Line 3,246: Line 2,979:
</table>
</table>
-
<p>
 
-
</p>
 
-
<p><h4>07/28/2014</h4></p>
 
-
<p>
+
</br><p><h4>07/28/2014</h4></p>
-
</p>
+
 
 +
 
<p>Collection pieces were quantified (minipreps made on 07/24/2014, see the Biosynthesis part for further details).
<p>Collection pieces were quantified (minipreps made on 07/24/2014, see the Biosynthesis part for further details).
</p>
</p>
-
<p>
+
 
-
</p>
+
<table style="width:300px">
<table style="width:300px">
Line 3,273: Line 3,003:
</table>
</table>
-
<p>
+
 
-
</p>
+
<p>The following table shows ligation details of the trichome promoter:
<p>The following table shows ligation details of the trichome promoter:
</p>
</p>
-
<p>
+
 
-
</p>
+
<table style="width:300px">
<table style="width:300px">
Line 3,316: Line 3,044:
</table>
</table>
-
<p>
 
-
</p>
 
-
<p><h4>07/29/2014</h4></p>
 
-
<p>
+
</br><p><h4>07/29/2014</h4></p>
-
</p>
+
 
 +
 
<p>Trichome Promoter() transformation
<p>Trichome Promoter() transformation
</p>
</p>
-
<p>
+
 
-
</p>
+
<p>Using an electrocompetent E. coli strain (DH5alpha) and 1.5 ul ligation (PTrich:GFP:TNos in 2alpha2), the mix is electroporated at 1500 V. Then, 300 uL of SOC are added and stored at 37 ºC with agitation.  
<p>Using an electrocompetent E. coli strain (DH5alpha) and 1.5 ul ligation (PTrich:GFP:TNos in 2alpha2), the mix is electroporated at 1500 V. Then, 300 uL of SOC are added and stored at 37 ºC with agitation.  
</p>
</p>
-
<p>
+
 
-
</p>
+
</body>
</body>
</html>
</html>

Revision as of 16:06, 1 August 2014

igem_logo



Biosynthesis


06/09/2014

The enzymes involved in the biosynthesis pathways are AtrD11, HarFAR, FAO, EaDAcT.

  • Codon optimization
  • Inner restriction sites eliminations by finding synonymous mutations
  • Addition of GB endings

06/10/2014

Codes for IDT known. MEGAGEM2014 - 25% off one order, up to $800

GBlocks designed to be compatible with biobricks and GoldenBraid (GB).


06/11/2014

We ordered the following gBlocks and primers.

  • EaDAcT: Eunymus alatus (adapted for GB) 1127 bp
  • HarFAR: Helicoverpa armigera (adapted for GB) 1400 bp
  • AtrD11: Amyelois transitella (order primers for GB) 1000 bp
    • I14Jun03 AtrD11 F1
    • I14Jun04 AtrD11 R1
  • FAO1: N.benthamiana primers
    • I14Jun01 FAO1 F1
    • I14Jun02 FAO1 R1
NameSequenceLenghtTm (NTI)Tm (Phusion)
I14Jun01_FAO1_F1cgccgtctcgctcgaatggagaaaaagagccatcc3549.962.4
I14Jun02_FAO1_R1cgccgtctcgctcgaagcttatcttgagaatttgccttcttttatc4654.563.7
I14Jun03Atr_D11_F1gcgccgtctcgctcgaatggttcctaataag3154.565.3
I14Jun04Atr_D11_R1gcgccgtctcgctcgaagctcaacgtttc295769.1

06/24/2014

We thought which parts of the GB collection could we use.

Strategy 1:

  • pP35S, pT35s (x2)
  • pAtUbq10, pTAtHSP18.2

Strategy 2:

  • pP35S, pT35s
  • pP35s, pTAtHSP18.2
  • pAtUbq10, pTAtHSP18.2

Strategy 3:

  • pP35S, pT35s
  • pP35s, pTTctp
  • pAtUbq10, pTAtHSP18.2

Pieces to take from GB2.0 colection:

pDGB2alpha1GB0483Kan
pDGB2alpha2GB0484Kan
pP35sGB0030Amp
pT35sGB0036Amp
pAtUbq10GB0223Amp
pTAtHSP18.2GB0035Amp
pTTctpGB0081Amp
pUPDGB0317Amp

Later we will need:

pDGB2omega1GB0487Smp
pDGB2omega2GB0488Smp

Prepare plaques with antibiotics Kan, Spm, Amp


06/25/2014

Grow the selected pieces from the GB collection in liquid medium (performed in laminar air flow cabinet).


06/26/2014

Culture in agar Petri dish. 2 plaques: Amp and Kan.

Minipreps with EZNA Plasmid DNA MiniKit I.

Expected digestions:

pP35s GB0030NotIBuffer: Orange2981, 1105
pT35s GB0036NotIBuffer: Orange2981, 304
pAtUbq10 GB0223NotIBuffer: Orange2981, 714
pTAtHSP18.2 GB0035NotIBuffer: Orange2981, 328
pTTctp GB0081NotIBuffer: Orange2981, 487

Electrophoresis analysis.

We got the expected bands in all cases.


01/07/2014

AtrD11 amplification by PCR with primers that contain extra nucleotides to introduce them in the sequence.

We made a PCR amplification using the Atr∆11 gene as a template and the oligos: R +F

Reagents needed:

  • 32.5 uL of H2O miliQ
  • 10 uL HF buffer
  • 2 uL dNTPs
  • 2.5 uL Reverse primer
  • 2.5 uL Forward primer
  • 1 uL template (AtrD11 gene)
  • 0.5 uL phusion (polimerase)

PCR parameters: The annealing temperature was 60º C and the extension temperature was 65º C.

Electrophoresis performed to check the PCR product, which was expected to be around 1 kb.

pUPD ligation of EaDAct, HarFar and Atr∆11

Reagents needed for the reaction of ligation:

  • 1 uL pUPD
  • 1 uL PCR product/gblock product
  • 1.2 uL buffer 10x
  • 1 uL BsmBI
  • 1 uL T4 ligase
  • 6.8 uL H2O miliQ

Vfinal= 12 uL

Termocycler parameters: The ligase temperature was 16º C and the BsmBI temperature was 37º C.

As a result, there are obtained three different pUPD plasmids containing the genes EaDacT, HarFAR and Atr∆11.


07/02/2014

E. coli transformation

This step is performed in a laminar air flow cabinet (LAF).

We have used an electrocompetent E. coli strain (DH5alpha) and a sample from each product of ligation made in the previous step (three pUPD plasmids, each of them containing one of the three genes), so transformation is made three times.

Reagents needed:

  • E. coli aliquot
  • 1.5 uL of ligation in pUPD (for each gene: EaDacT, HarFAR, Atr∆11)

Each mix is introduced in a electroporation vial and electroporated at 1500 V, then 300 uL of SOC are added to each vial. All of them were incubated at 37 ºC for 1 hour.

After incubation, culture in Petri plates (always in a LAF).

2 cell-culture dishes per transformation (with Ampicillin), one with 50 uL and the other with the remaining volume.

Petri plates are incubated at 37 ºC for 16 h.


07/03/2014

Transformed colonies selection. The white ones are recultured in liquid medium. One colony of each transformation is picked and cultured in 3.5 mL LB and 7 uL Amp. This step is repeated three times:

  • 3x 1 colony of EaDacT in pUPD + LB + Amp
  • 3x 1 colony of HarFAR in pUPD + LB + Amp
  • 3x 1 colony of AtrD11 in pUPD + LB + Amp

All tubes are incubated at 37 ºC overnight in agitation.


07/04/2014

Digestions in silico using Vector NTI to check after minipreps if ligations are correct.

EaDacTNotI2981, 1167
RsaI1876, 1343, 532, 306, 91
HarFARNotI2981, 1440
PvuII2564, 1394, 463
AtrD11NotI2981, 1056
BanII2570, 803, 351, 314

Digestion reagents:

  • 0.5 uL restriction enzyme
  • 2.5 uL buffer
  • 21 uL H20 (miliQ)
  • 1 uL sample

Preparation of master mixes

  • Master mix for NotI
    • 5 uL NotI
    • 25 uL Orange
    • 210 uL H20
  • Master mix for RsaI
    • 1.5 uL RsaI
    • 7.5 uL Tango
    • 63 uL H20
  • Master mix for PvuII
    • 1.5 uL PvuII
    • 7.5 uL Green
    • 63 uL H20
  • Master mix for BanII
    • 1.5 uL BanII
    • 7.5 uL Tango
    • 63 uL H20

Perform electrophoresis to check if the size of the fragments from the digestions is correct.

Comments:

  • We picked blue colonies instead of white by mistake. We need to pick colonies again but this time make sure we pick white colonies.
  • For the repetition we must find another enzyme instead of BanII as we found out that it doesn't cut very well.

07/06/2014

We picked again 3 colonies for each construction, and we made sure that we picked the WHITE ones. We cultivated them in a "double check" (name invented by us) liquid medium. Those tubes contain:

  • 3.5 mL LB
  • 7 uL Amp
  • 7 uL X-Gal
  • 3.5 uL IPTG (turns the tube blue if the colonies picked were blue)

07/07/2014

We made minipreps of yesterday's culture. Thanks to our "double check" medium we found which colonies were well picked. Finally we had minipreps of tubes HarFAR 1, 2, 3; EaDAcT 3 and AtrD11 2, 3.

Once we had the minipreps, we perform the digestions to check which were correct and send them to sequencing. This time we selected RsaI instead of BanII. The in silico digestions were as follows.

EaDacTNotI2981, 1167
RsaI1876, 1343, 532, 306, 91
HarFARNotI2981, 1440
PvuII2564, 1394, 463
AtrD11NotI2981, 1056
RsaI1879, 1310, 467, 327, 54

Preparation of master mixes

  • Master mix for NotI
    • 3.5 uL NotI
    • 17.5 uL Orange
    • 147 uL H20
  • Master mix for RsaI
    • 2 uL RsaI
    • 10 uL Tango
    • 84 uL H20
  • Master mix for PvuII
    • 2 uL PvuII
    • 10 uL Green
    • 84 uL H20

We run the electrophoresis gel to check if this time we have done it correctly.

Everything was OK. We sent AtrD11 (3), HarFAR (3) and EaDAcT (3) to sequence.


07/08/2014

Now, while we wait for sequencing results, we go on as they were going to be correct in order to save time.

The next step is to build a transciptional unit (TU) with our sequences. A transcriptional unit is a structure composed by promoter, coding sequence (CDS) and terminator in an alpha or omega vector.

Reagents needed for ligation:

  • 1 uL promoter 75 ng/uL
  • 1 uL terminator 75 ng/uL
  • 1 uL CDS 75 ng/uL
  • 1 uL vector alpha
  • 1.2 uL ligase buffer 10x
  • 1 uL T4
  • 1 uL BsaI
  • 4.2 uL H20

Total: 12 uL

Take into account that if we want to make binary constructions later (merge 2 TU in a same vector), we need to clone each TU in a different alpha vector.

Strategy Promoter-Terminator:

AtrD11P35sT35s40.41
HarFARP35sTatHSP39.68
EaDAcTPAtUbqTatHSP32.27

Adjust concentrations to 75 ng/uL for ligation reaction

Initial concentrations (nanodrop):

PieceConcentrationsVolumeVolume of H20 to add
PAtUpb442.6 ng/uL34 uL166.6 uL
pTatHSP235.4 ng/uL36 uL77 uL
T35s194.9 ng/uL37.5 uL60 uL
P35s454.7 ng/uL36 uL182 uL
2alpha157.1 ng/uL-We will need to put 1.5 uL of this one
2alpha2104.0 ng/uL38 uL14.7 uL
AtrD11359.3 ng/uL20 uL75.8 uL
HarFAR404.4 ng/uL15 uL65.9 uL
EaDAcT155.6 ng/uL10 uL10.7 uL

Ligation reaction

  • P35s:AtrD11:T35s in 2alpha1
    • 1 uL P35s
    • 1 uL T35s
    • 1 uL AtrD11
    • 1.5 uL 2alpha1
    • 1.2 uL ligase buffer 10x
    • 1 uL T4
    • 1 uL BsaI
    • 3.7 uL H20
  • P35s:HarFAR:TatHSP in 2alpha2
    • 1 uL P35s
    • 1 uL TatHSP
    • 1 uL HarFAR
    • 1 uL 2alpha2
    • 1.2 uL ligase buffer 10x
    • 1 uL T4
    • 1 uL BsaI
    • 4.2 uL H20
  • PAtUbq:EaDAcT:TatHSP in 2alpha2
    • 1 uL PAtUbq
    • 1 uL TatHSP
    • 1 uL EaDAcT
    • 1 uL 2alpha2
    • 1.2 uL ligase buffer 10x
    • 1 uL T4
    • 1 uL BsaI
    • 4.2 uL H20

07/09/2014

Transformation of constructions in E. coli

We finally got the sequencing results from 07/07/2014.

  • Mutation in AtrD11 -> We threw away the colonies and transformed cells. We picked again white colonies.
  • HarFAR -> Sequencing correct
  • EaDAcT -> Synonim mutation in 601 (A -> T). This is a gBlock!

We took vectors 2omega1 (GB0487) and 2omega2 (GB0488) parts from the GB colection.

  • Worked in the LAF
  • Cultivated in a Petri dish with Spm
  • Let them grow for one day

Cultivate transformated cells in two Kan plaques (Kan matches vector alpha)

  • 50 mL transformation in one plaque
  • Rest of the culture in another (250 uL aprox)
  • Let them grow for one day

07/10/2014

Pick colonies and grow them in liquid medium.

  • 6 from AtrD11 (repetition because of mutation)
    • 3.5 mL LB
    • 7 uL Amp
    • 7 uL X-gal
    • 3.5 uL IPTG
  • 1 colony from 2omega1
    • 3.5 mL LB
    • 7 uL Spm
  • 1 colony from 2omega2
    • 3.5 mL LB
    • 7 uL Spm
  • 3 colonies from P35s:HarFAR:TatHSP
    • 3.5 mL LB
    • 7 uL Kan
  • 3 colonies from PAtUbq:EaDAcT:TatHSP
    • 3.5 mL LB
    • 7 uL Kan

Grow at 37 ºC in agitation overnight.

We have checked the promoters and terminators are both compatible with GB and BioBricks.

Only P35s and T35s work for both. pPnos could also work.

Pick 3 colonies of P35s:HarFAR:THsp and PAtUbq:EaDAcT:THsp. Culture in liquid medium with Kan.


07/11/2014

We made minipreps of yesterday's liquid culture. Thanks to our "double check" medium we found which colonies were well picked. Finally we had minipreps of tubes AtrD11 3 and 6; 2omega1; 2omega2; constructions P35s:HarFAR:TatHSP 1, 2, 3 and PAtUbq:EaDAcT:TatHSP 1, 2, 3.

Additionally, we have cultured each of the colonies named above to store them.


07/14/2014

We tested the minipreps made last friday by digestion. Once they were checked, we send the correct ones to sequencing. The in silico digestions were as follows.

PartsRetriction enzymeExpected Bands
PAtUbq:EaDacT:TatHSP in 2alpha2HindIII6322, 1722, 736, 221
P35s:HarFAR:TatHSP in 2 alpha2HindIII6322, 1794, 221
AtrD11NotI2961, 1056
2omega1BamHI6652, 382, 239
2omega2EcoRV6652, 621

Preparation of master mixes

  • Master mix for HindIII
    • 3.5 uL HindIII
    • 17.5 uL Red
    • 147 uL H20
  • Master mix for NotI
    • 1.5 uL NotI
    • 7.5 uL Orange
    • 63 uL H20
  • Mix for EcoRV
    • 0.5 uL EcoRV
    • 2.5 uL Red
    • 21 uL H20
  • Mix for BamHI
    • 0.5 uL PvuII
    • 2.5 uL Green
    • 21 uL H20

We run the electrophoresis gel to check if this time we have done it correctly.

Everything was OK except the AtrD11 (3), which had some partial digestion. It was the reason we sent AtrD11 (6) to sequence.


07/15/2014

We got the sequencing results from yesterday and everything was OK, so we made the transcriptional units ligation.

Reagents needed for the reaction of ligation (Total volume = 12 uL):

  • P35s:AtrD11:T35s in 2alpha1
    • 1 uL P35s
    • 1 uL T35s
    • 1 uL AtrD11
    • 1.5 uL 2alpha1
    • 1.2 uL ligase buffer 10x
    • 1 uL T4
    • 1 uL BsaI
    • 3.7 uL H20
  • P35s:HarFAR:T35s in 2alpha2
    • 1 uL P35s
    • 1 uL T35s
    • 1 uL HarFAR
    • 1 uL 2alpha2
    • 1.2 uL ligase buffer 10x
    • 1 uL T4
    • 1 uL BsaI
    • 4.2 uL H20
  • P35s:EaDAcT:T35s in 2alpha2
    • 1 uL P35s
    • 1 uL T35s
    • 1 uL EaDAcT
    • 1 uL 2alpha2
    • 1.2 uL ligase buffer 10x
    • 1 uL T4
    • 1 uL BsaI
    • 4.2 uL H20

Note: Concentrations were previously adjusted to 75 ng/uL. Only the AtrD11 was adjusted from 250.2 ng/uL.

Finally, we prepared liquid cultures in order to store in glicerol. The tubes we used and their respective antibiotics were:

  • Amp
    • pAtrD11 (6)
    • pEaDAcT (3)
    • pHarFAR (3)
  • Kan
    • P35:HarFAR:TatHSP in 2alpha2 (3)
    • PPAtUbq:EaDAcT:TatHSP in 2apha2 (3)

07/16/2014

Storage in glycerol of the HarFAR (GB1018), AtrD11 (GB1019), EaDAcT (GB1020), P35s:HarFAR:TatHSP in 2alpha2 (GB1021) and PAtUbq:EaDAcT:TatHSP in 2alpha2 (GB1022) . we made 3 tubes: one for us, one for the GB collenction and one for reserve.

The procedure is to mix 700 uL of culture with 300 uL of glycerol 50%, spin it and store it in the -80ºC.


07/17/2014

Pick 3 colonies of P35s:AtrD11:T35s, P35s:HarFAR:T35s and P35s:EaDAcT:T35s. Culture in liquid medium with Kan.

Digestions in silico.

Transcriptional unitsRestriction enzymesExpected bands
P35s:AtrD11:T35sEcoRI6323, 2269
NcoI390, 8202
P35s:HarFAR:T35sHindIII933, 6322, 1722
NcoI8587, 390
P35s:EaDAcT:T35sHindIII6322, 2366
EcoRV683, 8021

Preparation of reagents needed for genomic extraction of Candida tropicalis for FAO1.


07/18/2014

Mistake in P35s:AtrD11:T35s, P35s:HarFAR:T35s and P35s:EaDAcT:T35s minipreps. Repeat tomorrow.


07/19/2014

Minipreps of P35s:AtrD11:T35s, P35s:HarFAR:T35s and P35s:EaDAcT:T35s. Concentration measuments with nanodrop.

Transcriptional unit DNA concentration (ng/uL)
P35s:AtrD11:T35s (1)164 ng/uL
P35s:AtrD11:T35s (2)168 ng/uL
P35s:AtrD11:T35s (3)147.4 ng/uL
P35s:HarFAR:T35s (1)125.3 ng/uL
P35s:HarFAR:T35s (2)114.5 ng/uL
P35s:HarFAR:T35s (3)140.3 ng/uL
P35s:EaDAcT:T35s (1)144.2 ng/uL
P35s:EaDAcT:T35s (2)137.9 ng/uL
P35s:EaDAcT:T35s (3)128.5 ng/uL
Stuffer fragment135.5 ng/uL
2omega1196.8 ng/uL
2omega2175.4 ng/uL

Digestions of P35s:AtrD11:T35s, P35s:HarFAR:T35s and P35s:EaDAcT:T35s and gel electrophoresis to check if transciptional units have been assembled OK.

All digestions were correct except P35s:EaDAcT:T35s (2).

Ligation in omega vectors.

  • P35s:AtrD11:T35s + P35s:HarFAR:T35s in 2omega1
    • 1 uL P35s:AtrD11:T35s (75 ng/uL)
    • 1 uL P35s:HarFAR:T35s (75 ng/uL)
    • 1 uL 2omega1 (75 ng/uL)
    • 1 uL BsmBI (5-10 ud)
    • 1 uL T4 ligase (5-10 ud)
    • 1 uL buffer ligase (3 ud)
    • 4 uL H20
  • P35s:EaDAcT:T35s in 2omega2
    • 1 uL stuffer fragment (75 ng/uL)
    • 1 uL P35s:EaDAcT:T35s (75 ng/uL)
    • 1 uL 2omega2 (75 ng/uL)
    • 1 uL BsmBI (5-10 ud)
    • 1 uL T4 ligase (5-10 ud)
    • 1 uL buffer ligase (3 ud)
    • 4 uL H20

Set the reaction: 25 cycles x (37ºC 2 min, 16ºC 5 min).

Omega vectors include a resistance to spectinomycin.


07/20/2014

Transform and grow in Petri dishes yesterday's ligations: P35S:AtrD11:T35S + P35S:HarFAR:T35S in 2omega1 and P35S:EaDAcT:T35S in 2omega2.


07/21/2014

Pick colonies of P35S:AtrD11:T35S + P35S:HarFAR:T35S in 2omega1 (3) and P35S:EaDAcT:T35S in 2omega2 (2).


07/22/2014

We made minipreps of yesterday's liquid culture. Selected tubes:

  • P35S:AtrD11:T35S + P35S:HarFAR:T35S in 2omega1(Tubes 1, 2 and 3)
  • P35S:EaDAcT:T35S in 2omega2 (Tubes 1 and 2)

Additionally, we have cultured each of the colonies named above in order to store them.

Digestions in silico made to check the transcriptional units:

Transcriptional unitsRestriction enzymeExpected bands
P35S:AtrD11:T35S + P35S:HarFAR:T35S in 2omega1EcoRV9307, 2251
BamHI6652, 4906
P35S:EaDAcT:T35S in 2omega2EcoRV6652, 1044, 817, 683
NcoI8806, 390

Digestion master mixes:

  • Master mix for NotI
    • 1.5 uL NotI
    • 7.5 uL Orange buffer
    • 63 uL H20
  • Master mix for NcoI
    • 1.5 uL NcoI
    • 7.5 uL Tango buffer
    • 63 uL H20
  • Master mix for BamHI
    • 2 uL BamHI
    • 10 uL Green buffer
    • 84 uL H20
  • Master mix for EcoRV
    • 4 uL EcoRV
    • 20 uL Red buffer
    • 168 uL H20

Note: Trichome promoter digestion preparation included.

All digestions were correct except the transcriptional unit of EaDAct in 2omega2 (P35s:EaDAcT:T35S).

*Aquí falta la imagen de las digestiones 22/07

Miniprep quantification:

PieceTubeConcentration (ng/uL)Volume (uL)
Trichome promoter in pUPD1317.126
Trichome promoter in pUPD3354.832
P35S:EaDAcT:T35S in 2omega21350.733
P35S:EaDAcT:T35S in 2omega22271.133
P35S:AtrD11:T35S + P35S:HarFAR:T35S in 2omega11306.331
P35S:AtrD11:T35S + P35S:HarFAR:T35S in 2omega12296.628
P35S:AtrD11:T35S + P35S:HarFAR:T35S in 2omega13246.033

All of the pieces named above were adjusted at 75 ng/uL.

Piece Tube numberFinal Volume (uLVolume to be added (uL)
Trichome promoter in pUPD1109.9383.93
Trichome promoter in pUPD3151.40119.4
P35S:EaDAcT:T35S in 2omega21154.30121.3
P35S:EaDAcT:T35S in 2omega22119.3086.30
P35S:AtrD11:T35S + P35S:HarFAR:T35S in 2omega11126.6095.60
P35S:AtrD11:T35S + P35S:HarFAR:T35S in 2omega12110.7082.70
P35S:AtrD11:T35S + P35S:HarFAR:T35S in 2omega13108.2475.20

As the digestions of the transcriptional unit (TU) of EaDAcT were incorrect, we repeated the process from the ligation step.

We transformed the same TU in a E. coli competent strain (DH5alpha). Then, the transformants were cultured in LB media and Spm and stored at 37 ºC overnight.

Pieces taken from the GoldenBraid 2.0 collection were cultured in solid growth media:

With Amp:

  • pTNos (GB0037)
  • pGFP (GB0059)
  • pLuciferase (GB0096)

With Kan:

  • P35S:Rosea:TNos
  • TA29:Barnase:TNos (GoldenBraid 1.0 piece)

Finally, in order to obtain the FAO1 gene, we want to extract the Candida tropicalis genome, so we have picked a colony of C. tropicalis. To check the extraction protocol, we used a yeast previously tested, Saccharomyces cerevisiae. We have cultured C. tropicalis in YPD media and S. cerevisiae in YPDA media at 28 ºC (5 mL).


07/23/2014

Candida genome extraction

Cultures measured at 600 nm:

  • S. cerevisiae Abs = 1.07
  • C. tropicalis Abs = 0.39

S. cerevisiae is recultured with new media (1:2) because the previous media was saturated. 2.25 mL of YPD media were mixed with 2.25 mL of S. cerevisiae culture. The mix has to grow at 28 ºC until the exponential phase is reached.

The absorbance was measured again:

  • S. cerevisiae Abs = 0.52
  • C. tropicalis Abs = 0.40

Buffers needed for the genome extraction were prepared freshly.The genome of both strains of yeast were extracted following the protocol:

  • Grow yeast in 2 or 5 mL YPD to exponential phase.
  • Collect cells in 1.5 mL eppendorf-cup (centrifugation 20 s, 6000 rpm).
  • Wash once with 1 mL sterile water.
  • Resuspend cells in 200 uL protoplast-buffer (100 mM Tris-HCl, pH 7.5, 10 mM EDTA, 1000 units Zymolyase/mL, 10 uL beta-mercaptoethanol/mL; prepare freshly!). Incubate at 37ºC for 1-2 h and finally resuspend by turning the cups.
  • Add 200 uL of Lysis-Mix (0.2 M NaOH, 1% SDS) an mix carefully (Don't vortex!).
  • Incubate at 65 ºC for 20 min and cool inmediately on ice.
  • Add 200 uL of 5 M KAc (pH 5.4) and mix carefully (Don't vortex!) and incubate 15 min on ice.
  • Centrifuge (13,000 rpm, 3 min) and transfer supernatant in a fresh cup.
  • Add 2 uL RNase A (10 mg/mL) and incubate at 37 ºC for 30 min.
  • Add 600 uL isopropanol and mix carefully (Don't vortex!). Incubate at room temperature for 5 min ad centrifuge (13,000 rpm, 30 s).
  • Remove the supernatant and wash with 70% ethanol (10 min at room temperature).
  • Centrifuge (13,000 rpm, 30 s) and remove the supernatant.
  • Dry DNA pellet in a speed-vacuum (not longer than 3 min!) and resuspend in 50 uL TE-buffer.
  • Store chromosomal DNA at 4 ºC (Don't freeze!). Concentration and quality can be checked in an agarose gel (loading 1/10 of the volume).

Genomic quantification:

OrganismConcentration
S. cerevisiae72.2 ng/uL
C. tropicalis1397.1 ng/uL

Electrophoresis made for check the extraction quality was not correct.

* Añadir imagen electroforesis extracción genomas candida y saccharomyces (23/07)

EaDAcT and collection pieces made yesterday were recultured in liquid media.


07/24/2014

The genomic quality of both organisms (C. tropicalis and S. cerevisiae) was checked in an agarose gel:

*Atención: incluir aquí el gel de geómico de candida y cerevisiae del 24/07

We got the Candida genome band, however, the Saccharomyces genome band was not present.

Additionally, minipreps of the liquid culture made yesterday were made and also recultured in solid agar plate.

  • P35S:EaDAcT:T35S in 2omega2 (tubes 1, 2, 3)
  • Collection pieces:
    • pTNos (GB0037)
    • pGFP (GB0059)
    • pLuciferase (GB0096)
    • P35:Rosea:TNos
    • TA29:Barnase:TNos

Miniprep checking is going to be done tomorrow.


07/25/2014

Digestions in silico made for checking yesterday's minipreps:

Pieces/TURestriction enzymeExpected bands
P35S:EaDAcT:T35SEcoRV6652, 1044, 817, 683
NcoI8806, 390
pTNosNotI2981, 570
pGFPNotI2981, 795
pLuciferaseNotI2981, 1731
P35S:Rosea:TNosBglII2495, 2302
NcoI4407, 390
TA29:Barnase:TNosBglII2825, 2245

Digestion master mixes:

  • Master mix for NotI
    • 2 uL NotI
    • 10 uL Orange buffer
    • 84 uL H20
  • Master mix for NcoI
    • 2 uL NcoI
    • 10 uL Tango buffer
    • 84 uL H20
  • Master mix for BglII
    • 2 uL BglII
    • 10 uL Orange buffer
    • 84 uL H20
  • Master mix for EcoRV
    • 1.5 uL EcoRV
    • 7.5 uL Red buffer
    • 63 uL H20

An agarose gel was made to check the transcriptional unit and the other pieces:

*¡Atención! Aquí falta un gel (grande) con el resulado de las digestiones de las piezas de la colección y de la Ea

All pieces were correct except the TU corresponding to P35:EaDAcT:T35S.


07/28/2014

Once the Candida tropicalis genome DNA is obtained, the FAO1 gene can be amplified by PCR.

PCR reaction reagents:

ReactionReagentVolume (uL)
FAO1-PCR1Template0.5
Buffer HF (5X)10.0
dNTPs2.0
Oligo R (JUL06)2.5
Oligo F (JUL05)2.5
Phusion polymerase0.5
H2O32.0
FAO1-PCR2Template0.5
Buffer HF (5X)10.0
dNTPs2.0
Oligo R (JUL08)2.5
Oligo F (JUL07)2.5
Phusion polymerase0.5
H2O32.0

Annealing temperatures

  • FAO1-PCR1: 59 ºC
  • FAO1-PCR2: 64 ºC

PCR products were checked using an electrophoresis. Expected bands:

  • FAO1-PCR1: 1157 bp
  • FAO1-PCR2: 1015 bp

Both FAO1 PCR products were not correct.

As the EaDAcT TU was not correct, ligation reaction was done again. The following table shows ligation details:

ReagentVolume (uL)
Trichome promoter1
GFP1
TNos1
BsaI1
p2alpha21
T4 ligase1
Ligase buffer1
H2O3
Total Volume10

07/29/2014

As the FAO1 PCR was not correct, we repeated the reaction. Below is a table showing the details:

ReagentFAO1-PCR1FAO1-PCR2
Template (C. tropicalis genome)(uL)2.52.5
HF Buffer (uL)30.030.0
dNTPs (uL)1010.010.0
Oligo R (uL)12.512.5
Oligo F (uL)12.512.5
Phusion polymerase (uL)1.51.5
H2O (uL)181.0181.0

PCR temperatures, 25 cycles:

StepTemperature (ºC)Time
Initialization982 min
Denaturation9820 s
Annealing50, 55, 60, 65
Extension7245 s
Final elongation727 min

We made a mistake preparing the FAO1-PCR1 adding the wrong template, so we do not expect the correct FAO11-PCR1 product.

EaDAcT Transcriptional Unit (TU) transformation

Using an electrocompetent E. coli strain (DH5alpha) and 1.5 ul ligation (P35S:EaDAcT:T35S in 2omega2), the mix is electroporated at 1500 V. Then, 300 uL of SOC are added and stored at 37 ºC with agitation.


07/30/2014



Switch


07/04/2014

Adquisition of S. cerevisiae genomic DNA. (5 uL, stored in the fridge)


07/28/2014

We had the genome of S. cerevisiae, needed to extract the target genes that are going to be used to build the switch. However we finally used our genome extraction (see Biosynthesis part, date 07/23/2014 for further details).

Previously we have designed a cupple of primers to amplify the CUP1 and CUP2 genes present in the yeast.

PCR reaction reagents:

ReagentCUP1-PCR1CUP2-PCR2
Template0.5 uL0.5 uL
Buffer HF (5X)10.0 uL10.0 uL
dNTPs2.0 uL2.0 uL
Oligo R (JUL06)2.5 uL2.5 uL
Oligo F (JUL05)2.5 uL2.5 uL
Phusion polymerase0.5 uL0.5 uL
H2O32.0 uL32.0 uL

Annealing temperature: both 61 ºC

PCR products were checked using an electrophoresis. Expected bands:

  • CUP1-PCR1: 386 bp
  • CUP2-PCR2: 348 bp

Both CUP1 and CUP2 PCR products were correct.



Trichome promoter


07/03/2014

Genomic DNA extraction from Nicotiana tabacum. We need the genome of this organism because we want to obtain the trichome promoter from the NtCPS2 gene.

  • Obtain 100 mg of the tobacco leaves (5 disks made with a 1.5 mL vial). Made it twice.
  • Introduce the disks inside the tube.
  • Introduce the two tubes in liquid nitrogen.
  • Remove them from the liquid nitrogen and store at -80ºC until use.
  • Remove one tube from -80ºC and re-introduce them in liquid nitrogen.
  • Grind the disks.
  • Add 600 uL of CTAB (2%) buffer (pre-heat at 65ºC.)
  • Grind the mixture.
  • Add RNAse (1.6 uL at M = 100 ug/uL for each mL of CTAB buffer).
  • Vortex it and maintain at 65ºC for 45 min. Mix it by inversion 5-15 min.
  • Add 600 uL cloroform:isoamilic alcohol. Vortex it.
  • Centrifuge 15 min at 13000 rpm (or 10 min at 14500 rpm.
  • Recover the supernatant by aspiration (with a 200 uL pipet).
  • Repeat the last three steps.
  • Add one volume o isopropanol and mix well by inversion (10 times).
  • To precipitate, maintain 20 min on ice or at -80ºC during 5 min.
  • Centrifuge 10 min at 13000 rpm (4ºC).
  • Discard the supernatant by decantation (be carefull with the pellet).
  • Wash with 600 uL ethanol (80%).
  • Centrifuge 5 min at 13000 rpm.
  • Discard the ethanol by pipeting and let it dry a few minutes.
  • Resuspend it in 50-100 uL H2O miliQ or with TE buffer.
  • Store at -20ºC.

Measurement of genomic concentration with nanodrop.

  • Tabacco 1: 182 ng/uL (Thrown away)
  • Tabacco 2: 620 ng/uL (Stored at -20ºC)

Electrophoresis performed to check the genomic size of tobacco (to see if it is degradated).

It is correct.


07/10/2014

PCR of genomic extraction of tobacco in order to amplify the trichome promoter CPS2.

Ordered primers

  • IGEMJULO1
  • IGEMJULO2

Ajust primers to a 100 uM concentration:

  • IGEMJUL01 + 566 uL miliQ H2O
  • IGEMJUL02 + 691 uL miliQ H2O

Use a 1:10 alicuot for PCR (10 uM).

Reagents needed for PCR:

  • 0.5 uL template
  • 10 uL buffer HF 5x
  • 2 uL dNTPs
  • 2.5 uL oligo R
  • 2.5 uL oligo F
  • 0.5 uL Pfu
  • 32 uL miliQ H2O

Final volume: 50 uL

Parameters:

  • 98 ºC (2 min)
  • 35 cycles
    • 98 ºC (10 sec)
    • 59 ºC (15 sec)
    • 72 ºC (45 sec)
  • 72 ºC (7 min)

We didn't get PCR product.


07/11/2014

Repeat PCR with different parameters.

1 2 3 4 5

Template 0.5 0.5 0.5 0.5 0.5

Buffer (5x) 0.5 0.5 0.5 0.5 0.5

dNTPs 2 2 2 2 2

Oligo R 2.5 2.5 2.5 2.5 2.5

Oligo F 2.5 2.5 2.5 2.5 2.5

Phu 0.5 0.5 0.5 0.5 0.5

Buffer 32 32 32 32 32

1, 2 and 5 contain buffer F; 3 and 4 contain buffer GC.

PCR parameters

  • 98 ºC (2 min)
  • 35 cycles
    • 98 ºC (10 sec)
    • 1, 3, 5 -> 59 ºC (15 sec). 2, 4 -> 55 ºC (15 sec)
    • 72 ºC (45 sec)
  • 72 ºC (7 min)

No PCR products again.

Repeat PCR again with other parameters.

Buffer HF Buffer GC

Template 2 2

Buffer (5x) 40 40

dNTPs 8 8

Oligo R 10 10

Oligo F 10 10

Phu 2 2

Buffer 128 128

Set 4 tubes with each buffer at different temperatures: 49, 52, 55, 60.

  • 98 ºC (2 min)
  • 35 cycles
    • 98 ºC (10 sec)
    • 49, 52, 55, 60 ºC (15 sec)
    • 72 ºC (45 sec)
  • 72 ºC (7 min)

No PCR products again.


07/14/2014

Repeat PCR again with more genomic.

Buffer HF Buffer GC
Template55
Buffer (5x)5050
dNTPs1010
Oligo R12.512.5
Oligo F12.512.5
Phu2.52.5
Buffer107.5107.5

Same parameters as before except annealing temperatures which are: 50, 53, 57, 59 ºC.

We still without having any amplification.


07/18/2014

Repeat the PCR with other enzyme.

  • 12.5 uL Q5 Master mix (2x).
  • 1.25 uL forward primer 10 uM
  • 1.25 uL reverse primer 10 uM
  • 0.5 uL template 620 ng/uL
  • 9.5 uL H2O

Set 4 reactions at 50, 53, 55, 59 ºC.

  • 98 ºC (30 sec)
  • 35 cycles
    • 98 ºC (10 sec)
    • 50, 53, 55, 59 ºC (15 sec)
    • 72 ºC (45 sec)
  • 72 ºC (2 min)

The DNA fragment of interest is around 1.5 kb so we see we finally obtained amplification at 55 and 59 ºC reactions.


07/19/2014

Trichome promoter PCR product ligation in pUPD.

  • 1 uL pUPD
  • 1 uL PCR product
  • 1 uL BsmBI (5-10 ud)
  • 1 uL T4 ligase (5-10 ud)
  • 1.2 uL buffer ligase (3 ud)
  • 6.8 uL H20

Set the reaction: 25 cycles x (37ºC 2 min, 16ºC 5 min).


07/20/2014

Transform and grow in Petri dishes yesterday's ligation of the trichome promoter in pUPD.


07/21/2014

We picked colonies of the trichome promoter in pUPD and grown it in liquid culture.


07/22/2014

We made minipreps of yesterday's liquid culture. Additionally, we have recultured them in solid growth media.

Digestions in silico made to check the insertion:

PieceRestriction enzymeExpected bands
Trichome Promoter in pUPDNotI2981, 1523
EcoRV3942, 562

Note: To see further details of digestion master mixes, go to the biosynthesis part, date 07/22/2014.


07/23/2014

Yesterday's digestions were correct, so the trichome promoter in pUPD was send to sequencing.


07/24/2014

Results of sequencing the promoter were obtained:

MutationPosition
T insertion?
T insertion?

07/28/2014

Collection pieces were quantified (minipreps made on 07/24/2014, see the Biosynthesis part for further details).

PieceConcentration (ng/uL)Initial Volume (uL)Final Volume (uL)
GFP318.835148.8
TNos 400.835186.5

The following table shows ligation details of the trichome promoter:

ReagentVolume (uL)
Trichome promoter1
GFP1
TNos1
BsaI1
p2alpha21
T4 ligase1
Ligase buffer1
H2O3
Total Volume10

07/29/2014

Trichome Promoter() transformation

Using an electrocompetent E. coli strain (DH5alpha) and 1.5 ul ligation (PTrich:GFP:TNos in 2alpha2), the mix is electroporated at 1500 V. Then, 300 uL of SOC are added and stored at 37 ºC with agitation.