Team:UC Chile/Cyanolux/Biobricks

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<h1>Constructs detail</h1>
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<h1>Plasmid backbones</h1>
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<h2>pSB1C3_IntK (BBa_K743006)</h2>
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Neutral recombination plasmid backbone. Kanamycin resistance as a selectable marker for integration. EcoRI restriction site in RS2. Plasmid backbone designed for Gibson Assembly.
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<html><img src="https://static.igem.org/mediawiki/2012/d/d0/UC_Chile-C1.1.jpg" align="right" width="800"><p style="width : 200px;">
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[[File:UC Chile-IntKplasmidResult.jpg | 400px ]]
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Integrative plasmid designed for the expression of RFP under synechocystis psbAB promoter also with a kanamycin resistance cassette for selection in cyanobacteria. The construct is flanked by neutral recombination sites RS1 and RS2, homologous to synechocystis chromosome.</p></html>
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<h2>pSB1C3_IntS (BBa_K743010)</h2>
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Suceptibility generating plasmid backbone. Spectinomycin resistance as a selectable marker for integration. mRFP1 under constitutive promoter in E.coli for negative selection of standard assembly.
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[[File:UC Chile-IntSplasmidResult.jpg | 600px ]]
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<h1>Biobricks and Constructs</h1>
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<h2>BBa_K743000</h2>
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First recombination site of IntK plasmid backbone.
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<html><img src="https://static.igem.org/mediawiki/2012/b/b9/UC_Chile-C1.2.jpg" align="right" width="800"><p style="width : 200px;">
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[[File:UC Chile-BBa_K743000.jpg | 230px ]]
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C1.2
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This integrative plasmid is designed for the expression of RFP under synechocystis psaA2 promoter with a kanamycin resistance cassette for selection in cyanobacteria. The construct is flanked by neutral recombination sites RS1 and RS2, homologous to synechocystis chromosome.
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<h2>BBa_K743001</h2>
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Second recombination site of IntK plasmid backbone. Illegal EcoRI restriction site present. As it always is used as a downstream part, illegal site does not interfere with Standard Assembly.
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[[File:UC Chile-BBa_K743001.jpg | 250px ]]
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<h2>BBa_K743002</h2>
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<i>Synechocystis</i> PCC. 6803 Citocrome C promoter.
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[[File:UC Chile-BBa_K743002.jpg | 250px ]]
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<h2>BBa_K743009</h2>
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pSB1C3_IntK(BBa_K743006) with mRFP1 under <i>Synechocystis</i> PpsaAB promoter.
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<html><img src="https://static.igem.org/mediawiki/2012/4/48/UC_Chile-C2.1.jpg" align="right" width="800"><p style="width : 200px;">
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[[File:UC Chile-BBa_K743009.jpg | 500px ]]
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C2.1
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Construct designed from pPMQAK1 expression plasmid, which replicates both in E. coli and cyanobacteria and is biobrick-compatible. It codes for RFP under synechocystis psbAB promoter.</p></html>
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<h2>BBa_K743016</h2>
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pSB1C3_IntK(BBa_K743006) with mRFP1 under <i>Synechocystis</i> PpsbA2 promoter.
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[[File:UC Chile-BBa_K743016.jpg | 500px ]]
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<h2>BBa_K743014</h2>
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pSB1C3_IntK(BBa_K743006) with LuxAB genes from Photorhabdus luminescent under <i>Synechocystis</i> Transaldolase promoter.
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[[File:UC Chile-IntKplasmidLuxABxl.jpg | 600px ]]
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<h2>BBa_K743015</h2>
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pSB1C3_IntK(BBa_K743006) with LuxAB genes from Vibrio fisherii under <i>Synechocystis</i> Transaldolase promoter.
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[[File:UC Chile-IntKfullplasmidResult.jpg | 600px ]]
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<html><img src="https://static.igem.org/mediawiki/2012/c/c6/UC_Chile-C2.2.jpg" align="right" width="800"><p style="width : 200px;">
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C2.2
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Construct designed from pPMQAK1 expression plasmid, which replicates both in E. coli and cyanobacteria and is biobrick-compatible. It codes for RFP under synechocystis psaA2 promoter.</p></html>
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<h2>BBa_K743018</h2>
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pSB1C3_IntK(BBa_K743006) with sfGFP gene with rapid degradation tag under <i>Synechocystis</i> Transaldolase promoter.
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[[File:UC Chile-BBa_K743018sfGFPtag.jpg | 600px ]]
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<a href="https://2012.igem.org/Team:UC_Chile/Cyanolux/Notepad"><img src="https://static.igem.org/mediawiki/2012/a/ab/UC_Chile-Continue_button.jpg" align="right">
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<html><img src="https://2012.igem.org/File:UC_Chile-C3.1.jpg" align="right" width="800"><p style="width : 200px;">
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C3
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Construct designed from pPMQAK1 expression plasmid, which replicates both in E. coli and cyanobacteria and is biobrick-compatible. It codes for RFP under synechocystis psbAB promoter and cuts out the whole Ccdb toxin gene.</p></html>
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<html><img src="https://2012.igem.org/File:UC_Chile-C3.2.jpg" align="right" width="800"><p style="width : 200px;"></p></html>
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<html><img src="https://2012.igem.org/File:C4.jpg" align="right" width="800"><p style="width : 200px;">Expression plasmid for the expression of LuxA and LuxB genes under psbAB promoter.
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<html><img src="https://2012.igem.org/File:C5.jpg" align="right" width="800"><p style="width : 200px;">C5<br>
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Integrative plasmid designed to integrate LuxA and LuxB and a kanamycin resistance cassette downstream the 3' end of the transaldolase gene whose mRNA levels where seen to oscillate in a circadian manner peaking at hour 14, just after dusk. It is expect the same for LuxAB mRNA levels.
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Integrative plasmid designed to integrate LuxA and LuxB and a kanamycin resistance cassette downstream the 3' end of the transaldolase gene whose mRNA levels where seen to oscillate in a circadian manner peaking at hour 14, just after dusk. It is expect the same for LuxAB mRNA levels.</p>
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<p>C6<br>
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Starting from construct C1, we designed an integrative plasmid that codes for LuxA and LuxB under the genomic transaldolase promoter (Pta). This time we decided to use the reverse complement of kanamycin resistance gene so both LuxAB and the resistance gene end at a double terminator sequence. You can check the problems we had with the standard kanR-double terminator (P1003+B0014) in our lab notebook.</p>
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<p>C7.1<br>
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Starting from psb1A3_intC integration plamid (BB_K390300) kindly provided to us by USU igem 2010, we designed a construct composed of LuxCDEG operon under synechocystis sigE promoter, whose mRNA abundance has been seen to oscillate in a circadian manner, with a peak at hour 8, before dusk.</p>
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<p>C7.1<br>
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Starting from psb1A3_intC integration plamid (BB_K390300) kindly provided to us by USU igem 2010, we designed a construct composed of LuxCDEG operon under synechocystis caa3  gene promoter, whose mRNA abundance has been seen to oscillate in a circadian manner, with a peak at hour 8, before dusk.</p>
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[[File:UC_Chile-PSB1C3_INTK.jpg|center]]
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[[File:UC_Chile-PS81C3_INTS.jpg|center]]
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== Brief description of characterized biobricks utilized ==
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== Biobricks ==
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Latest revision as of 09:55, 26 October 2012

Project: Luxilla - Pontificia Universidad Católica de Chile, iGEM 2012