Team:PennState/diauxie/conclusions

Diauxie Elimination

Introduction

The System

Strategies

Progress

Conclusions

Parts

References

NHR Biosensors

NHR Introduction

Phthalate Biosensor

BPA Biosensor

Even though we have created the xylose inducible constructs with the GFP reporter, further testing is in progress with the newly created E. coli cell lines. There are eight different tests that could be performed to show the effects of sugar induction, xylR production, and xylE production. The tests previously performed were mostly in DH5α E. coli cells which contain the entire xyl operon in the chromosome. This means that without our constructs in the cell, xylose would be naturally metabolized. The fluorescence testing results would reflect the xylose metabolism from the natural behavior instead of the inserted plasmid.

The W3110 ∆xylB-G cells have no xylose metabolism and should not grow at all with xylose. The fluorescence results should show altering levels with xylose induction since there is still xylR present. The W3110 ∆xylB-R cells should show no xylose induction with any of the promoters since there will be no xylR protein present. Both of these cells will be tested with xylR constitutive production on the plasmid, xylE constitutive production, or a combination of both. These tests should show different fluorescence strengths for sugar induction. The strongest effect should occur with xylR and xylE under control of the constitutive promoter on the plasmid, in the W3110 ∆xylB-R cells.

We hope to see more of a difference in glucose-xylose induction between the natural promoter and our engineered promoters. Because we are increasing the strength of the RNA polymerase binding site to resemble a constitutive promoter sequence, there is the possibility that XylR binding will have no effect on our system. We are hoping that XylR interacts with the polymerase to strengthen its binding, therefore causing xylose to positively regulate transcription. If this turns out to not be the case than we have a third strategy that involves using the xyl transcriptional control region from Bacillus bacteria. In this system XylR acts as a repressor, only xylose binding to XylR allows transcription to take place. We would put a constitutive promoter upstream of this region causing the genes to be expressed only in the presence of xylose.