Team:PennState/diauxie/conclusions

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.

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.

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.

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.

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.

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.

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 <td valign="top" id="pagecontent" width="80%"><span style="font-size: 16pt">Conclusions</span>
 <hr/>
-<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 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.</p>
+<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>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.  </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>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.</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>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>