Team:Groningen/Template/MODULE/Notebook/vector/week1

From 2014.igem.org

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Making BioBrick compatible vectors pIL253 (<a href="http://parts.igem.org/Part:BBa_K1365301">BBa_K1365301</a>) and pNZ8048g with the <i>mrfp</i> (<a href="http://parts.igem.org/Part:BBa_J04450">BBa_J04450</a>) insert
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<b>goal</b>: Isolate <i>mrfp</i> insert out of pSB1C3 and ligate in pIL253 and pNZ8048g to make biobrick compatible vectors for <i>L. lactis</i>
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<i>mrfp</i> was primed out of pSB1C3 by primers designed for pIL253 and pNZ8048g
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<div class="item"><i>mrfp</i> was primed out of pSB1C3 by primers designed for pIL253 and pNZ8048g</div>
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<div class="item">The reaction mixtures were checked on gel, this yielded in some positive results for the constructs for both pIL253 and pNZ8048g</div>
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<div class="item">The vectors pIL253 and pNZ8048g were digested in such a way that the <i>mrfp</i> constructs will fit in</div>
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<div class="item">The <i>mrfp</i> inserts were digested with the same or complementary enzymes as the vectors</div>
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<div class="item">The backbones and <i>mrfp</i> inserts were checked on a gel</div>
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<div class="item">The construct were checked on gel, but did not yield any positive results for the pNZ8048g with <i>mrfp</i></div>
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<div class="item">Instead of <i>mrfp</i> (<a href="http://parts.igem.org/Part:BBa_J04450">BBa_J04450</a>), the gene for the purple-blue chromoprotein amilCP (<a href="http://parts.igem.org/Part: BBa_K592025">BBa_K592025</a>), and a construct containing the promoter CP29 (<a href="http://parts.igem.org/Part:BBa_K1033222">BBa_K1033222</a>) with RBS (<a href="http://parts.igem.org/Part:BBa_B0034">BBa_B0034</a>) and the Superfolded GFP gene (<a href="http://parts.igem.org/Part:BBa_K1365020">BBa_K1365020</a>) were primed with the same primers used for the <i>mrfp</i> for pIL253 and pNZ8048g</div>
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<div class="item">The constructs were positively checked on gel</div>
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<div class="item">The <i>mrfp</i>, the gene for the purple-blue chromoprotein amilCP and the construct containing the promoter CP29 with RBS and the Superfolded GFP gene were ligated with pIL253 and transformed to <i>L. lactis</i> NZ9000
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<div class="item">The the gene for the purple-blue chromoprotein amilCP and the construct containing the promoter CP29 with RBS and the Superfolded GFP gene inserts were also ligated with pNZ8048g and transformed <i>L. lactis</i> NZ9000</div></div>
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<div class="item">The transformants of pIL253 were plated on M17 agar with erythromycin and the transformants of pNZ8048g were on M17 agar with chloramphenicol</div>
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<div class="item">No colonies were yielded from these transformations</div>
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The reactions were loaded on a gel and this yielded positive results for the constructs for both pIL253 and pNZ8048g
 
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The backbones pIL253 and pNZ8048g are digested so that the <i>mrfp</i> constructs will fit in them
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Also the <i>mrfp</i> inserts are digested with the same or complementary enzymes
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The backbones and <i>mrfp</i> inserts are checked on a gel
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The gel did not yield good results for the pNZ <i>mrfp</i>
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Instead of <i>mrfp</i>, <i>amil-cp</i> and <i>cp29-rbs-sfgfp</i> were primed with the same primers used for <i>mrfp</i> for pIL253 and pNZ8048g
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This was run on a gel and yielded the right results
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The <i>mrfp</i>, <i>amil-cp</i> and <i>cp29-rbs-sfgfp</i> inserts for pIL253 were ligated to pIL253 and transformed to <i>L. lactis</i> NZ9000 and the <i>amil-cp</i> and <i>cp29-rbs-sfgfp</i> inserts for pNZ8048g were also ligated to pNZ8048g and transformed <i>L. lactis</i> NZ9000
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The transformants of pIL253 were plated on M17 agar with erythromycin and the transformants of pNZ8048g were on M17 agar with chloramphenicol
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This transformation did not yield any colonies
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Revision as of 20:53, 17 October 2014

September 29 - October 5
 
Making BioBrick compatible vectors pIL253 (BBa_K1365301) and pNZ8048g with the mrfp (BBa_J04450) insert
 
mrfp was primed out of pSB1C3 by primers designed for pIL253 and pNZ8048g
The reaction mixtures were checked on gel, this yielded in some positive results for the constructs for both pIL253 and pNZ8048g
The vectors pIL253 and pNZ8048g were digested in such a way that the mrfp constructs will fit in
The mrfp inserts were digested with the same or complementary enzymes as the vectors
The backbones and mrfp inserts were checked on a gel
The construct were checked on gel, but did not yield any positive results for the pNZ8048g with mrfp
Instead of mrfp (BBa_J04450), the gene for the purple-blue chromoprotein amilCP (BBa_K592025), and a construct containing the promoter CP29 (BBa_K1033222) with RBS (BBa_B0034) and the Superfolded GFP gene (BBa_K1365020) were primed with the same primers used for the mrfp for pIL253 and pNZ8048g
The constructs were positively checked on gel
The mrfp, the gene for the purple-blue chromoprotein amilCP and the construct containing the promoter CP29 with RBS and the Superfolded GFP gene were ligated with pIL253 and transformed to L. lactis NZ9000
The the gene for the purple-blue chromoprotein amilCP and the construct containing the promoter CP29 with RBS and the Superfolded GFP gene inserts were also ligated with pNZ8048g and transformed L. lactis NZ9000
The transformants of pIL253 were plated on M17 agar with erythromycin and the transformants of pNZ8048g were on M17 agar with chloramphenicol
No colonies were yielded from these transformations
 
 
 
 
 

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