Team:PennState/diauxie/Strategies

We explored two options to make a glucose-independent, xylose-inducible system: 1) via the protein CRP*, and 2) by selectively engineering the promoter region sequence.

The first strategy was the main focus of the 2007 Penn State iGEM team. This approach used 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. The problem with this approach is that CRP* is not specific to the xyl operon; it also regulates transcription in many other locations. Expressing CRP* in the cell can lead to having other systems turned on or off irregularly, possibly having toxic consequences.

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 CRP 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 RNA polymerase binding. Depending on the mechanism(s) of interaction between XylR and RNA polymerase components, too strong of a promoter may result in high levels of constitutive expression (in the absence of xylose), which is undesirable.

Xyl Operon

Four different altered promoter regions were ordered from GeneArt. Promoter “PN” was synthesized as the wild-type promoter sequence with biobrick ends. This promoter is necessary for testing comparisons with altered region promoters. Promoter P1 was ordered as the natural promoter region with a scrambled CRP binding site. The P1 promoter should show weak RNA polymerase binding and transcription and will help to further elucidate the roles of CRP and XylR in xyl promoter expression. The “medium strength” promoter P3 contained a scrambled CRP binding site along with an altered RNA polymerase binding site, the latter changed to be a combination of the wild-type and consensus binding sequences. Finally promoter P5 contains a scrambled CRP binding site and an RNA polymerase binding site matching the consensus sequence.

To test the right-facing promoters we cloned RBS/GFP (E0240) downstream of each sequence. Fluorescence readings were used to measure the levels of induction.

@@ Line 161: / Line 161: @@
   <h4><acronym title="Nuclear Hormone Receptor">NHR Biosensors</acronym><br/></h4>
   <dl id="denav">
-   <dd><a href="hbintro" title="Intro to Endocrine Disruption, pseudoestrogens, pthalates, nuclear hormone receptors, and our goals">Introduction</a></dd>
+   <dd><a href="https://2008.igem.org/Team:PennState/NHR/introduction">NHR Introduction</a></dd>
    <dd><a href="https://2008.igem.org/Team:PennState/smartfold/overview">Phthalate Biosensor</a></dd>
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 <td valign="top" id="pagecontent" width="80%"><span style="font-size: 16pt">Strategies</span>
 <hr/>
-<p class="start">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.</p>
+<p class="start">We explored two options to make a glucose-independent, xylose-inducible system: 1) via the protein CRP*, and 2) by selectively engineering the promoter region sequence.</p>
-<p>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.</p>
+<p>The first strategy was the main focus of the 2007 Penn State iGEM team.  This approach used 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. The problem with this approach is that CRP* is not specific to the xyl operon; it also regulates transcription in many other locations. Expressing CRP* in the cell can lead to having other systems turned on or off irregularly, possibly having toxic consequences.</p>
-<p>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.</p>
+<p>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 CRP 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 <em>E. coli</em> RNA polymerase binding. Depending on the mechanism(s) of interaction between XylR and RNA polymerase components, too strong of a promoter may result in high levels of constitutive expression (in the absence of xylose), which is undesirable.</p>
-<p>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.</p>
+<h4>Xyl Operon</h4>
+<img src="https://static.igem.org/mediawiki/2008/b/b5/Xyl_operon_penn_state.jpg" alt="[Plasmid Map]" title="Xyl Operon" style="width: 100%; border: solid 1px #000" />
+<p>Four different altered promoter regions were ordered from GeneArt.  Promoter “PN” was synthesized as the wild-type promoter sequence with biobrick ends.  This promoter is necessary for testing comparisons with altered region promoters.  Promoter P1 was ordered as the natural promoter region with a scrambled CRP binding site.  The P1 promoter should show weak RNA polymerase binding and transcription and  will help to further elucidate the roles of CRP and XylR in xyl promoter expression.  The “medium strength” promoter P3 contained a scrambled CRP binding site along with an altered RNA polymerase binding site, the latter changed to be a combination of the wild-type and consensus binding sequences.  Finally promoter P5 contains a scrambled CRP binding site and an RNA polymerase binding site matching the consensus sequence.</p>
+<p>To test the right-facing promoters we cloned RBS/GFP (E0240) downstream of each sequence.  Fluorescence readings were used to measure the levels of induction. </p>
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