Team:PennState/diauxie/conclusions

We have constructed the xylose-inducible constructs with the GFP reporter and testing is still in progress with newly constructed E. coli cell strains. Eight different tests are being performed to show the effects of sugar induction, XylR expression, and XylE expression. The initial tests were mostly performed with strain DH5α , which contains the entire xyl operon in the chromosome. Xylose metabolism in this strain thus poses a problem during the characterization of our constructs.

Newly constructed strains constitutively express xylR and xylE genes. Intracellular levels of XylR may be too low to accommodate the high copy plasmid used in our studies, and the presence of glucose may alter native XylR expression levels, thereby artificially lowering induction levels. Native xylose transport genes are also subject to glucose repression, so XylE will be independently expressed at high levels to ensure xylose uptake in the presence of glucose. Strain W3110 ∆xylB-G carries deletion in xylose metabolism (xylA and xylB) and transport (xylFGH) genes, and therefore would not cause any unwanted depletion of the xylose during testing of the promoter constructs. The level of induction from the test constructs with and without XylR production will be compared to evaluate the role of XylR levels on induction. Similarly, the effect of having XylE as the sole transporter will be studied. Strain W3110 ∆xylB-R is similar to the ∆xylB-G strain but also does not express XylR, and will serve to further study the role of XylR (no induction should be observed in the absence of XylR).

The W3110 ∆xylB-G cell strain alone will not exhibit any xylose metabolism or transport, and therefore would not cause any unwanted depletion of the xylose available for activation of the test constructs. The level of induction from the test constructs with and without XylR production will be compared to see the significance of XylR levels on induction. Similarly the effect of having XylE as the sole transport mechanism will also be observed.

Understanding the behaviour of these constructs will provide valuable insights into the natural control region and its dependence on both xylose transport and XylR concentration for induction. These studies will also help us further characterize our engineered promoter, and to find the conditions where glucose/xylose diauxie is eliminated.

One of our hypotheses is that XylR interacts with RNA polymerase to strengthen its binding. Xylose-bound XylR should positively regulate transcription, and the hope is that a strengthened promoter sequence will still require activation by XylR (and not just increase constitutivity). If this turns out not to be the case, the we have proposed a third strategy involving the xyl transcriptional control region from Bacillus sp. In this system XylR (which is very different from the XylR produced by E. coli) is known to act as a repressor, such that xylose binding to XylR derepresses and enables RNA polymerase binding and transcriptional activation. The “diauxie” phenotype that occurs for xyl expression in Bacillus is due to the ability of glucose (or glucose phosphate) to directly interact with XylR and influence its repressor functions. To eliminate glucose sensitivity, we are engineering the Bacillus XylR regulatory protein to act independently of glucose or other metabolites resulting from glucose metabolism.

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 <td valign="top" id="pagecontent" width="80%"><span style="font-size: 16pt">Conclusions</span>
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-<p class="start">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 strains.  There are eight different tests that are being performed to show the effects of sugar induction, XylR expression, and XylE expression. The tests previously performed were mostly in DH5α E. coli which contain the entire xyl operon in the chromosome.  Having natural xylose metabolism genes expressed from the chromosome poses a problem during the characterization of our constructs.  The amount of sugars available for induction will differ between cells using the xylose for energy and the cells not using it.</p>
+<p class="start">We have constructed the xylose-inducible constructs with the GFP reporter and testing is still in progress with newly constructed E. coli cell strains.  Eight different tests are being performed to show the effects of sugar induction, XylR expression, and XylE expression. The initial tests were mostly performed with strain DH5α , which contains the entire xyl operon in the chromosome.  Xylose metabolism in this strain thus poses a problem during the characterization of our constructs. </p>
-<p>The new test construct we are building contains constitutively expressed xylR and xylE genes. We believe that the levels of XylR available for activation are too low to accommodate our high copy plasmid, therefore artificially lowering the induction levels. XylE will also be expressed at higher levels so that lack of xylose transport will not inhibit transcription. As mentioned before XylE is a passive transport protein and depends on a concentration gradient of xylose, having this in place of the active XylFGH transport will allow a more gradual entry of xylose into the cell.</p>
+<p>Newly constructed strains constitutively express xylR and xylE genes. Intracellular levels of XylR may be too low to accommodate the high copy plasmid used in our studies, and the presence of glucose may alter native XylR expression levels, thereby artificially lowering induction levels. Native xylose transport genes are also subject to glucose repression, so XylE will be independently expressed at high levels to ensure xylose uptake in the presence of glucose. Strain W3110 ∆xylB-G carries deletion in xylose metabolism (xylA and xylB) and transport (xylFGH) genes, and therefore would not cause any unwanted depletion of the xylose during testing of the promoter constructs.  The level of induction from the test constructs with and without XylR production will be compared to evaluate the role of XylR levels on induction.  Similarly, the effect of having XylE as the sole transporter will be studied. Strain W3110 ∆xylB-R is similar to the ∆xylB-G strain but also does not express XylR, and will serve to further study the role of XylR (no induction should be observed in the absence of XylR). </p>
 <p>The W3110 ∆xylB-G cell strain alone will not exhibit any xylose metabolism or transport, and therefore would not cause any unwanted depletion of the xylose available for activation of the test constructs.  The level of induction from the test constructs with and without XylR production will be compared to see the significance of XylR levels on induction.  Similarly the effect of having XylE as the sole transport mechanism will also be observed. </p>
-<p>The W3110 ∆xylB-R should also show the significance of XylR from a different perspective.  Theoretically, no induction should be observed in the absence of XylR especially in the case of xylose without glucose.</p>
+<p>Understanding the behaviour of these constructs will provide valuable insights into the natural control region and its dependence on both xylose transport and XylR concentration for induction.  These studies will also help us further characterize our engineered promoter, and to find the conditions where glucose/xylose diauxie is eliminated. </p>
-<p>All these studies will provide valuable insight on the natural control region and its dependence on both xylose transport and XylR concentration for induction.  These studies will also help us further characterize our engineered promoter, and to find the conditions where glucose/xylose diauxie is eliminated. </p>
-<p>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 upstream 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. For diauxie elimination, we would put a constitutive promoter upstream of this region causing the genes to be expressed only in the presence of xylose.</p>
+<p>One of our hypotheses is that XylR interacts with RNA polymerase to strengthen its binding. Xylose-bound XylR should positively regulate transcription, and the hope is that a strengthened promoter sequence will still require activation by XylR (and not just increase constitutivity). If this turns out not to be the case, the we have proposed a third strategy involving the xyl transcriptional control region from Bacillus sp.  In this system XylR (which is very different from the XylR produced by E. coli) is known to act as a repressor, such that xylose binding to XylR derepresses and enables RNA polymerase binding and transcriptional activation. The “diauxie” phenotype that occurs for xyl expression in Bacillus is due to the ability of glucose (or glucose phosphate) to directly interact with XylR and influence its repressor functions. To eliminate glucose sensitivity, we are engineering the Bacillus XylR regulatory protein to act independently of glucose or other metabolites resulting from glucose metabolism.</p>