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

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8 – 12 September
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September 15 - September 19
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<b>Goal:</b> analysis of the possible made Biobricks.
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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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Because of the very low amount of gblocks given, a decision was made to do a PCR directly on the product itself, therefor multiplying it exponentially.
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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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All the O/N cultures were mini-prepped. The possible plasmids were cut with EcoRI and SpeI. After running on gel, the result showed us that 20 of the 40 clones contained all the Biobricks, though with a low yield of plasmids.
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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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A new strategy of assembling has been prepared and primers for this strategy have been made.  
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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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All the Biobricks were send for sequencing.  
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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 20:36, 17 October 2014

September 15 - September 19
 
goal: obtain the anti Staphylococcus aureus (p2s) and the Pseudomonas aeruginosa(pLASs)
 
A decision was made to assemble the P2s  pLASs with gibson assembly.  An RBS is downstream attached of P2, dspB and pLAS with tail PCR.
 
Afterwards a gibson tail was attached with pcr to p2+rbs, dspB+rbs, aiiA and double terminator. Then 59ng of p2, 400ng  of ssUSPDspB45, 65ng of nisA and 50ng of Double terminator and incubated for1:30 hours at 50 degrees celcius.
 
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.
 
The p2s system was verified through restriction analysis and sequencing.
 
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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