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Biobricks Submitted




The LEA fragment we used was kindly sent by Liu (?) and consists of the protein PM2 (LEA3) from soybean (Glycine max (L) Merr.). It was inserted into the pET28a vector and transformed into E. coli BL21 Star cells. In order to amplificate it and clone it into the pSB1C3, we used the forward primer actagtagcggccgctgcagATGGCGTCCAAGAAAC and the reverse primer tctagaagcggccgcgaattcTGCGTCTATATATAC (capital letters indicate the region of the sequence that pairs with the coding sequence of PM2).


The switch is formed by two different parts: the activator and the reporter. The activator part is a construction of two fragments: the NM domains of the protein Sup35, which confers to the protein the prionic activity, and the GR526 portion, which contains the DNA-binding and transcription-activation domains. The ligand-binding domain of the protein GR was eliminated, decoupling the response of the protein to the presence of glucocorticoids, and thus generating a constitutive transcription activation factor. The normal activity of this protein results in the activation of the genes preceded by the GRE (Glucocorticoid Response Element). When exposed to heat shock or other stress conditions, the NM domains start the prionic activity, eventually inhibiting the activation of transcription. This part was amplified by using the primers indicated by Lindquist et al (?), together with the sequence recommended to use for the ligation protocol with the plasmid pSB1C3. Those primers are: forward actagtagcggccgctgcagATGTCGGATTCAAAC and reverse tctagaagcggccgcgaattcTCCTGCAGTGGCTTG (again, capital letters represent the region that pairs with the coding sequence of NMGR526). The reporter part consists of the GRE and the reporter gene. In our experiments, we used LacZ as a reporter. The amplification of this part could not be made because we had some trouble finding the sequence of the GRE.