Team:PennState/diauxie/implementation

Diauxie Elimination

Introduction

Implementation

Progress

Parts

References

Hormone Biosensors

Introduction

Smart Fold

Overview

Parts

References

Nuclear Fusion

Overview

Parts

References

The system

In wildtype E. coli the xyl operon controls xylose metabolism. The genes xylAB code for xylose isomerase and kinase, and the genes xylFGH code for active xylose transport proteins. Another protein called XylE, a passive xylose transporter, exists elsewhere in the chromosome. A bidirectional operator controls transcription in both the direction of xylAB and xylFGH. Transcription is initiated when the protein XylR binds to xylose which is followed by binding to the XylR binding site located at both ends of the operator. The binding of CRP-cAMP to a single CRP binding site is also required for activation. cAMP is an indicator of the glucose concentration in the cell and only becomes present when glucose is depleted.

In can be seen that both the presence of xylose and the absence of glucose are naturally required for transcriptional activation, our goal was to make only the presence of xylose necessary.

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 year's 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 (BBa_E0240) downstream of each sequence. Fluorescence readings were used to measure the level of induction.