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

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8 – 12 September
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September 29 - October 5
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<b>Goal:</b> analysis of the possible made Biobricks.
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Obtaining the anti-<i>Pseudomonas aeruginosa</i> system with Three-A assembly
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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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Extra amounts of <i>dspB</i>, ssUSP45DspB with His-tag, <i>aiiA</i>, ssUSP45aiiA with His-tag, <i>pLAS</i>, the RBS, the double terminator, pSB2k3 vector, pSB1C3 vector and pSB1A3 vector were miniprepped to be used for the assembly
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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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<i>aiiA</i>, <i>dspB</i>, ssUSP45DspB with His-tag and ssUSP45aiiA with His-tag were digested with XbaI and PstI, and the RBS was cut with EcoRI and SpeI
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A new strategy of assembling has been prepared and primers for this strategy have been made.  
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These were eventually ligated for 3 hours into pSB2K3 and transformed into <i>E. coli</i> NEB cells
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<th>Name assembly</th><th>Upstream part</th><th>Downstream part</th><th>Backbone</th>
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<td>A1’</td><td>RBS</td><td><i>aiiA</i></td><td>pSB2K3</td>
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<td>A2’</td><td>RBS</td><td><i>dspB</i></td><td>pSB2K3</td>
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<td>A3’</td><td>RBS</td><td><i>dspB+HIS</i></td><td>pSB2K3</td>
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<td>A4’</td><td>RBS</td><td><i>aiiA+HIS</i></td><td>pSB2K3</td>
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After growing them for 1 day, colony PCR was done on the correct clones, these were inoculated for plasmid extraction
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All the Biobricks were send for sequencing.  
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Then A1’, A4’ was digested with XbaI and PstI. A2’ and A3’ was cut with EcoRI and SpeI. Then ligated
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<th>Name assembly</th><th>Upstream part</th><th>Downstream part</th><th>Backbone</th>
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<td>A1”</td><td>RBS + <i>aiiA</i></td><td>Double terminator</td><td>pSB1A3</td>
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<td>A2”</td><td><i>pLAS</i></td><td>RBS + <i>dspB</i></td><td>pSB1A3</td>
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<td>A3”</td><td><i>pLAS</i></td><td>RBS + <i>dspB+HIS</i></td><td>pSB1A3</td>
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<td>A4”</td><td>RBS + <i>aiiA+HIS</i></td><td>Double terminator</td><td>pSB1A3</td>
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After doing growing analyzing and miniprepping the correct clones, A1” and A3”was cut with EcoRI and SpeI. A2” and A4” was cut with XbaI and PstI and ligated afterwards
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<th>Name assembly</th><th>Upstream part</th><th>Downstream part</th><th>Backbone</th>
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<td>A1”’</td><td><i>pLAS</i>+RBS+<i>dspB</i></td><td>RBS+<i>aiiA</i>+Dterm</td><td>pSB1C3</td>
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<td>A2”’</td><td><i>pLAS</i>+RBS+<i>dspB+HIS</i>></td><td>RBS+<i>aiiA+HIS</i>+Dterm</td><td>pSB1C3</td>
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After transformation, the plasmids were isolated, and confirmed by sequencing
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Latest revision as of 00:46, 18 October 2014

September 29 - October 5
 
Obtaining the anti-Pseudomonas aeruginosa system with Three-A assembly
 
Extra amounts of dspB, ssUSP45DspB with His-tag, aiiA, ssUSP45aiiA with His-tag, pLAS, the RBS, the double terminator, pSB2k3 vector, pSB1C3 vector and pSB1A3 vector were miniprepped to be used for the assembly
 
aiiA, dspB, ssUSP45DspB with His-tag and ssUSP45aiiA with His-tag were digested with XbaI and PstI, and the RBS was cut with EcoRI and SpeI
 
These were eventually ligated for 3 hours into pSB2K3 and transformed into E. coli NEB cells
 
Name assemblyUpstream partDownstream partBackbone
A1’RBSaiiApSB2K3
A2’RBSdspBpSB2K3
A3’RBSdspB+HISpSB2K3
A4’RBSaiiA+HISpSB2K3
 
After growing them for 1 day, colony PCR was done on the correct clones, these were inoculated for plasmid extraction
 
Then A1’, A4’ was digested with XbaI and PstI. A2’ and A3’ was cut with EcoRI and SpeI. Then ligated
 
Name assemblyUpstream partDownstream partBackbone
A1”RBS + aiiADouble terminatorpSB1A3
A2”pLASRBS + dspBpSB1A3
A3”pLASRBS + dspB+HISpSB1A3
A4”RBS + aiiA+HISDouble terminatorpSB1A3
 
After doing growing analyzing and miniprepping the correct clones, A1” and A3”was cut with EcoRI and SpeI. A2” and A4” was cut with XbaI and PstI and ligated afterwards
 
Name assemblyUpstream partDownstream partBackbone
A1”’pLAS+RBS+dspBRBS+aiiA+DtermpSB1C3
A2”’pLAS+RBS+dspB+HIS>RBS+aiiA+HIS+DtermpSB1C3
 
After transformation, the plasmids were isolated, and confirmed by sequencing
 
 
 
 

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