Team:Groningen/Template/MODULE/Notebook/secretion/week9

From 2014.igem.org

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September 15 - September 19
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September 22 - September 28
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<b>goal:</b> obtain the anti <i>Staphylococcus aureus</i> (p2s) and the <i>Pseudomonas aeruginosa</i>(pLASs)
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<b>goal:</b> obtain the anti <i>Pseudomonas aeruginosa/i> system
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A decision was made to assemble the P2s  pLASs with gibson assembly.  An RBS is downstream attached of <i>P2</i>, <i>dspB</i> and <i>pLAS</i> with tail PCR.
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This week tried everything to assembly this system with the help of Gibson assembly. Every try failed miserably and at the end of the week it was decided to assemble this system with 3-A assembly
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Afterwards a gibson tail was attached with pcr to <i>p2+rbs</i>, <i>dspB+rbs</i>, <i>aiiA</i> and double terminator.
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Then 59ng of <i>p2</i>, 400ng  of <i>ssUSPDspB45</i>, 65ng of <i>nisA</i> and 50ng of Double terminator and incubated for1:30 hours at 50 degrees celcius.
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The gibson fragment was amplified with PCR after 20 cycles. With help of gradient PCR, we found the exact temperature of annealing. The p2s was then cloned into pSB1C3 and sequenced accordingly.
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The p2s system was verified through restriction analysis and sequencing.
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For the pLASs 70ng of <i>pLAS</i>, 400ng of <i>dspB+RBS</i>, 290ng of <i>aiiA</i> and 50ng of double terminator. Though after amplifying the fragment and loading it on gel, only a 1200bp band could be found.
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Revision as of 20:41, 17 October 2014

September 22 - September 28
 
goal: obtain the anti Pseudomonas aeruginosa/i> system
 
This week tried everything to assembly this system with the help of Gibson assembly. Every try failed miserably and at the end of the week it was decided to assemble this system with 3-A assembly
 
 
 
 

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