Team:Groningen/Template/MODULE/Notebook/secretion/week4

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<b>goal:</b> assemble the promotors with RBS and GFP (BBa_E0040) with double terminator.
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Assembling the promoters P2 (<a href="http://parts.igem.org/Part:BBa_I746103">BBa_I746103</a>) and pLas (<a href="http://parts.igem.org/Part:BBa_K091117">BBa_K091117</a>) with RBS and the gene coding for GFP (<a href="http://parts.igem.org/Part:BBa_ E0040">BBa_ E0040</a>) with double terminator (<a href="http://parts.igem.org/Part:BBa_ B0015">BBa_ B0015</a>)
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Biobricks pLAS, P2, GFP, RBS and Double terminator were assembled accordingly to their place in the construct with 3A assembly, ligated, chemically transformed and inoculated on kanamycine agar plates.
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<div class="item">The Biobricks pLAS, P2, GFP, RBS and Double terminator were assembled using three-A assembly, the constructs were ligated, and transformed to <i>E. coli</i> DH5α</div>
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<div class="item">The transformations were plated on LB agar with kanamycin</div>
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<div class="item">All the colourless colonies were plated to fresh plates again, and grown overnight</div>
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<div class="item">The construct was checked by colony PCR, ending up with the following constructs: Ps with RBS, pLas with RBS, and the gene coding for GFP with double terminator</div>
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<div class="item">Another colony PCR was performed with Phusion polymerase this time, in order to produce a more clean product for a second assembly</div>
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<div class="item">The primers and the synthetic gene ssUSP45DspB (<a href="http://parts.igem.org/Part:BBa_K1365111">BBa_K1365111</a>) arrived</div>
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<div class="item">The <i>mrfp</i> insert was cut out of pSB1C3, run on gel, and the linearized vector was purfied out of the gel</div>
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<div class="item">ssUSP45DspB was ligated with pSB1C3, and transformed to <i>E. coli</i> DH5α</div>
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<div class="item">Few colonies were visible after growing the transformation mixture of LB agar plates with chloramphenicol</div>
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<div class="item">After digestion, the constructs of the few colonies were checked on gel, only one colony showed the right size for the insert</div>
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The O/N grown colorless clones were picked and grown on their own individual plate. Afterwards, colony PCR on all the colonies showed a positive result for assembled: P2+RBS, pLAS+RBS, GFP+Dterm.
 
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After the colony PCR, another one was done with phusion DNA polymerase, so that the product could be used for a second assembly with (P2+RBS)+(GFP+Dterm) and (pLAS+RBS)+(GFP+Dterm).
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Corresponging in only a few clones which gave negative results after colony PCR. Therefore, another assembly with the constructs should be done.
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Primers came in, preparing them accordingly.
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The synthetic gene ssUSP45DspB, which arrived from IDT, was prepared according to the protocol made by IDT.
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1µl of pSB1C3 was cut with EcoRI and PstI. The digestion mix ran on a agarose gel, after which gel purification of the linearized backbone occurred.
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70ng of vector was used to ligate ssUSP45DspB.
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The yield of transformants was very low after transforming the ligation mixture and growing the bacteria. Only one colony contained the ssUSP45DspB according to gel.
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Revision as of 22:42, 17 October 2014

August 11 - August 15
 
Assembling the promoters P2 (BBa_I746103) and pLas (BBa_K091117) with RBS and the gene coding for GFP (BBa_ E0040) with double terminator (BBa_ B0015)
 
The Biobricks pLAS, P2, GFP, RBS and Double terminator were assembled using three-A assembly, the constructs were ligated, and transformed to E. coli DH5α
The transformations were plated on LB agar with kanamycin
All the colourless colonies were plated to fresh plates again, and grown overnight
The construct was checked by colony PCR, ending up with the following constructs: Ps with RBS, pLas with RBS, and the gene coding for GFP with double terminator
Another colony PCR was performed with Phusion polymerase this time, in order to produce a more clean product for a second assembly
The primers and the synthetic gene ssUSP45DspB (BBa_K1365111) arrived
The mrfp insert was cut out of pSB1C3, run on gel, and the linearized vector was purfied out of the gel
ssUSP45DspB was ligated with pSB1C3, and transformed to E. coli DH5α
Few colonies were visible after growing the transformation mixture of LB agar plates with chloramphenicol
After digestion, the constructs of the few colonies were checked on gel, only one colony showed the right size for the insert
 
 
 
 
 

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