Team:PennState/diauxie/Strategies

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.

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.

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.

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.

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-<td valign="top" id="pagecontent" width="80%"><span style="font-size: 16pt">Introduction</span>
+<td valign="top" id="pagecontent" width="80%"><span style="font-size: 16pt">Strategies</span>
 <hr/>
-<p class="start">There are currently a only few dependable transcriptional induction systems that are readily available for use in <cite>E. coli</cite>  and other bacteria. Some common systems use proteins such as LacI and AraC to sense the level of sugars such as lactose and arabinose respectively. In both these cases the presence of glucose affects the amount of transcription promoted by each operator; in wild-type bacteria the genes downstream of these operators are involved with metabolism of the sugar associated with the control of that gene system. This phenomenon of preferential sugar usage for cell growth is called <strong>diauxie</strong>.</p>
+<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>In this project our goal is to introduce <strong>a new induction system</strong> that uses xylose as the inducer. To make this system unique, transcription will be activated in the presence of xylose regardless of the presence of glucose in the cell. This will create a novel and useful new way of initiating transcription that isn’t limited to cases where glucose is not a carbon source.</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>In addition to creating <strong>a valuable new tool for the part registry</strong>, this project has useful applications in the <a href="http://en.wikipedia.org/wiki/Bioconversion_of_biomass_to_mixed_alcohol_fuels">bioproduction</a> industry, including (but not limited to) conversion of biomass to ethanol using bacteria. Common biomass feedstocks used for production contain a 50:50 mixture of glucose and xylose. When cells are grown on this mixed carbon source, diauxie causes glucose to be used first, then depending on the process xylose is consumed or removed as waste. This leads to inefficiency in production, especially if a continuous process is desired. Separation of xylose from glucose is very costly, and so is the lag time when the cells are switching from glucose to xylose metabolism. <strong>The induction system we are creating would eliminate this problem by eliminating diaxuie, leading to more efficient production by using  both sugars at the same time.</strong></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>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>