Team:Groningen/Template/MODULE/Notebook/secretion/week8

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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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Obtaining the anti-<i>Staphylococcus aureus</i> system (p2s) and the anti-<i>Pseudomonas aeruginosa</i> system(pLASs)
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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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<div class="item">Both systems were assembled by using Gibson assembly</div>
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<div class="item">Downstream of the <i>pLas</i> a RBS is attached together with the promoter P2 and  <i>dspB</i> with tail PCR</div>
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<div class="item">A Gibson tail was attached with PCR to the P2 with RBS, <i>dspB</i> with RBS, <i>aiiA</i> and the double terminator </div>
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<div class="item">ssUSP45DspB, and <i>nisA</i> was added and incubated for 90 minutes at 50 °C</div>
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<div class="item">The Gibson fragment was amplified with gradient PCR</div>
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<div class="item">The construct was digested, checked on gel, and send for sequencing</div>
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<div class="item">Only 1200 base pairs of the construct could be found on gel</div>
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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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Revision as of 00:34, 18 October 2014

September 15 - September 19
 
Obtaining the anti-Staphylococcus aureus system (p2s) and the anti-Pseudomonas aeruginosa system(pLASs)
 
Both systems were assembled by using Gibson assembly
Downstream of the pLas a RBS is attached together with the promoter P2 and dspB with tail PCR
A Gibson tail was attached with PCR to the P2 with RBS, dspB with RBS, aiiA and the double terminator
ssUSP45DspB, and nisA was added and incubated for 90 minutes at 50 °C
The Gibson fragment was amplified with gradient PCR
The construct was digested, checked on gel, and send for sequencing
Only 1200 base pairs of the construct could be found on gel
 
 
For the pLASs 70ng of pLAS, 400ng of dspB+RBS, 290ng of aiiA 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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