Team:PennState/diauxie/Strategies

We explored two options to make a glucose independent, xylose inducible system: via the protein CRP*, and selectively engineering base changes in the promoter region sequence.

The first strategy was the main focus of the previous Penn State iGEM team. This approach was the use of a mutated version of the protein CRP called CRP* which acts as CRP bound to cAMP. This means that our system would always be turned on when xylose is present, even if glucose is also present because lower glucose levels correspond to higher levels of cAMP. The problem with this approach is CPR* is not specific to the xyl operon, it also regulates transcription in many other locations. Having lots of CRP* in the cell can lead to having other systems turned on or off irregularly, possibly being toxic to the cell.

This year we focused on constructing and characterizing engineered alterations of the xyl promoter region. The operator controlling genes for xylose metabolism are dependent on binding both the CRP-cAMP and XylR-xylose activated complexes. We attempted to engineer the promoter region so only XylR transcriptional control remained. To accomplish this, we first scrambled the CRP binding site DNA by random base changes. With this sequence deactivated, the possibility of CRP binding was eliminated; this is also significant because it may repress transcription when bound in the operator region without cAMP. The second alteration was to strengthen the RNA polymerase binding sites to compensate for the loss of CRP promotion. The binding affinity was strengthened by changing a few bases in the polymerase binding region. This was done in steps so that the regions began to resemble a consensus sequence for E. coli polymerase binding.

Four different altered promoter regions were ordered from GeneArt. The lowest level, we named PN, was synthesized as the natural promoter region with biobrick ends. This promoter is necessary for testing comparisons with altered region promoters. The second level promoter, P1, was ordered as the natural promoter region with a scrambled CRP binding site. The P1 promoter should show weak RNA polymerase binding but will help explain the effects of CRP binding for transcription. The medium strength, third promoter, P3, contained a scrambled CRP binding site along with the altered RNA polymerase binding site changed to be a combination of the wild type and consensus binding sequences. Finally, the strongest promoter, P5, was synthesized with a scrambled CRP binding site and an RNA polymerase binding site matching the consensus sequence.