Part:BBa_K909012:Experience

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Applications of BBa_K909012

Our team iGEM Wageningen UR 2014 used this BioBrick for the design of a toggle switch which can be induced by CI repressor (BBa_K1493703). We assembled this promoter with a gfp gene under Elowitz RBS (BBa_I13504) to construct the screening BioBrick BBa_K1493502. We used the L-Rhamnose mediated characterization method described on our wiki page for characterization of this promoter.

To do this the promoter with gfp was assembled to a L-rhamnose inducible promoter (pRha) regulating the CI repressor protein (BBa_K1493510) or a pRha regulating the LacI repressor protein (BBa_K149320) in pSB3K3. To indicate the concentration of repressor protein in the cell for each concentration of rhamnose, pRha with gfp is used (BBa_K1493501)(Figure 1).

Figure 1. Average GFP fluorescence of E. coli DH5α carrying a pSB3K3 plasmid containing the pRha gfp BioBrick. At t=0 the cells were induced with different concentrations of rhamnose in triplo. Fluorescence is measured in a plate reader in time (A) and at time point 8.25 (B) normalized for OD600.

We wanted to determine the RPU value of this promoter. Therefore, we used pTet (BBa_K149504) as a reference part.
Figure 2. A graph showing the relative fluorescence unit of pCI/Lac and pTet. Both E. coli DH5α strains containing BBa_K1493502 BBa_K1493504 were grown in M9 medium with 2% glycerol in absence of rhamnose in triplo. pCI/Lac shows an average RFU value of 6000 and pTet shows an average RFU of 8700 at time point 8.25h.

Figure 3. A) The average RFU values of E. coli NEB5α carrying a pSB3K3 plasmid containing the BioBricks pRha CIλ and pCI/lac gfp. B) The average RFU values of E. coli NEB5α carrying a pSB3K3 plasmid containing the BioBricks pRha lacI and pCI/Lac gfp. Cells were grown in M9 medium with 2% glycerol and induced with 0%, 0.001%, 0.01%, 0.05% or 0.2% rhamnose or 0.2% glucose at t=0. Fluorescence was measured over time and data of time point 8.25h are shown in the graphs. Rhamnose concentrations of 0.001% and 0.01% have no substantial effect on fluorescence, compared to 0% rhamnose. Cells grown in 0.05% and 0.2% rhamnose show a lower RFU value compared to 0% rhamnose indicating that the pCI/Lac is repressed by the repressor protein regulated by the rhamnose promoter. 0.2% glucose has an effect on the RFU, as the values are lower than 0% rhamnose.

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