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Team:Tuebingen/Results/Overview
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Revision as of 00:24, 5 October 2013
Cloning
This year, we succeeded in cloning all missing parts required for our measuring system. While mPR X.l., mPR D.r., Mig, Padh and Rox were available in pUC-IDT vector or pGEM T-easy vector from last years iGEM Team (LINK ZU ALTEM WIKI), Pfet, Panb1, Psuc2 were cloned from yeast genome. Furthermore we used the fluorescent reporter protein mOrange(BBa_E2050) and the transcriptional terminator Tadh(BBa_K801012) from the parts registry. A galactose inducible promotor(BBa_J63006) from the registry was used to create some plasmids which we want to use for the characterization of some of our parts. The cloning of luciferase succeeded only shortly before wiki freeze. As a result, the assemblies requiring luciferase were not performed anymore.
Assembly
Our general assembly strategy is illustrated in fig.1. The pRS vectors do not fit the RFC10 criteria and can therefore not be used the for 3A-Assembly. Instead we used the BioBrick RFC10 vector pTUM100 (BBa_K801000) which was kindly provided by the iGEM Team of the TU Munich to fuse promotor and coding sequence. pTUM100 is a high copy shuttle plasmid which is based on the commercially available pYES2 vector and contains the cyc1 transcription terminator after the BioBrick suffix. Thus we could use some of the pTUM constructs directly for characterization of our repressible promotors (Pfet-mOrange-pTUM100, Panb-mOrange-pTUM100, Psuc-mOrange-pTUM100, Padh-mOrange-pTUM100). The parts required for the next steps of characterization were equipped with a the transcriptional terminator Tadh by ligation into a pSB1C3 plasmid containing Tadh. The resulting assemblies were again BioBricks. These inserts were then ligated into the pRS shuttle vectors losing parts of their prefix and/or suffix. In total we have created 29 new plasmids!
