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Team:Groningen:Notebook:Backbone
From 2014.igem.org
Notebook
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Vector
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
All the remaining ligation mixtures for both the pIL253, and the pNZ8048g were stored at - 20 °C
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
For our system it was important that the Biobricks could be transformed to our chassis L. lactis. This meant that we could not use the standard backbones provided by iGEM. Here we describe how we synthesized a Biobrick compatible backbone for L. lactis.
October 6 - October 12
Two of the ligation mixtures stored at -20 °C, were transformed again to L. lactis (using more higher concentrations of ligation mixture this time), the mixture of mrfp with pIL253 vector, and the mixture of promoter CP29, with RBS and the Superfolded GFP gene with the pNZ8048g vector
This time the transformation with pIL253 was succesfull
The size of the construct was checked by colony PCR
