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Team:PennState/diauxie/parts
From 2008.igem.org
Strategies
We explored a few options to make a glucose independent, xylose inducible system: using the protein CRP*, and selectively engineering base changes in the promoter region sequence.
The first approach that week took was the use of a mutated version of the protein CRP called CRP*. This strategy was the main focus of last years Penn State iGEM team. CPR* acts as CRP bound to cAMP which means that our system would always be turned on when xylose is present, even if glucose is also present. The problem with this approach is that CPR* is not specific to the xyl operon; it regulates transcription in many other locations. Having lots of CRP* in the cell can lead to having other systems turned on or off when they shouldn’t be, possibly being toxic to the cell.
This year we focused on constructing and characterizing alterations of the xyl promoter region which we engineered. As mentioned before, the operator controlling genes for xylose metabolism is dependent on both the binding of CRP-cAMP, and XylR-xylose. We tried to engineer the promoter region such that only XylR control remained. To do this we scrambled the CRP binding site by making random base changes to this region. This was done to eliminate the possibility of CRP binding, especially because it is thought to possibly repress transcription when bound without cAMP. The RNA polymerase binding sites were also strengthened to compensate for the loss of CRP promotion. This was done by changing a few bases at a time in this region so that they more resembled a consensus sequence for E. coli polymerase binding.
Four different altered promoter regions were ordered from geneart. The lowest level (PN) was synthesized as the natural promoter region with biobrick ends. The second level (P1) was ordered as the natural promoter region with the CRP binding site scrambled. The third promoter (P3) contained a scrambled CRP binding site and the RNA polymerase binding site was changed to be a mix between the wild type and consensus sequence. Finally the strongest promoter (P5) was synthesized with a scrambled CRP binding site and an RNA polymerase binding site matching the consensus sequence.
To test each of these promoters we cloned GFP (E0240) downstream of each sequence. Fluorescence readings were used to measure the level of induction.
