Difference between revisions of "Part:BBa K4046000"
Line 4: Line 4:
  
 
This is a modified version of the transcriptional repression factor DhdR, which is responsive to the oncometabolite D-2-HG. DhdR is a transcriptional repression factor isolated from the bacteria <i> Achromobacter denitrificans </i>. The sequence was human codon optimized for expression in mammalian cells. Additionally, the sequence had a nuclear localization sequence attached to the end, for transport into the nucleus as part of our genetic reporter system, and a FLAG tag attached to the end.  
 
This is a modified version of the transcriptional repression factor DhdR, which is responsive to the oncometabolite D-2-HG. DhdR is a transcriptional repression factor isolated from the bacteria <i> Achromobacter denitrificans </i>. The sequence was human codon optimized for expression in mammalian cells. Additionally, the sequence had a nuclear localization sequence attached to the end, for transport into the nucleus as part of our genetic reporter system, and a FLAG tag attached to the end.  
−
 
−
A schematic for the operation of our system is shown below.
 
−
[[File:T--Duke--DhdrSchematic.png|600 px]] <br>
 
−
 
−
<b>Figure 1: This figure outlines the mechanism of the interaction between the DhdR allosteric transcription factor and the <i>dhdO</i> binding site.</b>
 
  
 
[[File:T--Duke--DhdR_gel.png|300 px]] <br>
 
[[File:T--Duke--DhdR_gel.png|300 px]] <br>
Line 14: Line 9:
  
 
As part of our goal to construct a biosensor system for the compound D-2-HG, we will utilize a plasmid expressing the DhdR gene to provide baseline expression of this repressor gene. In a <i> wild-type </i> environment, without the presence of DhdR, we expect normal expression of the reporter protein. However, when DhdR is present, it will bind to the DhdO binding site, allosterically blocking the transcription of our reporter gene. When D-2-HG is elevated, particularly in IDH1 mutant cells, it binds to DhdR, releasing it from the binding site. This allows for transcription of the downstream reporter protein sequence, resulting in brighter expression that is visible in our <i> in vivo </i> droplet system. Since D-2-HG levels are elevated due to the IDH1 mutation, we expect that there will be an increase in fluorescence or luminescence due to the release of the allosteric transcription factor caused by the binding of the upregulated oncometabolite. When we perform drug screening assays on our completed co-culture system, we will associate decreased fluorescence or luminescence with lower levels of D-2-HG, which is associated with a variety of downstream metabolic impacts.  
 
As part of our goal to construct a biosensor system for the compound D-2-HG, we will utilize a plasmid expressing the DhdR gene to provide baseline expression of this repressor gene. In a <i> wild-type </i> environment, without the presence of DhdR, we expect normal expression of the reporter protein. However, when DhdR is present, it will bind to the DhdO binding site, allosterically blocking the transcription of our reporter gene. When D-2-HG is elevated, particularly in IDH1 mutant cells, it binds to DhdR, releasing it from the binding site. This allows for transcription of the downstream reporter protein sequence, resulting in brighter expression that is visible in our <i> in vivo </i> droplet system. Since D-2-HG levels are elevated due to the IDH1 mutation, we expect that there will be an increase in fluorescence or luminescence due to the release of the allosteric transcription factor caused by the binding of the upregulated oncometabolite. When we perform drug screening assays on our completed co-culture system, we will associate decreased fluorescence or luminescence with lower levels of D-2-HG, which is associated with a variety of downstream metabolic impacts.  
 +
 +
A schematic for the operation of our system is shown below.
 +
[[File:T--Duke--DhdrSchematic.png|600 px]] <br>
 +
 +
<b><font size="+1">Figure 1: This figure outlines the mechanism of the interaction between the DhdR allosteric transcription factor and the <i>dhdO</i> binding site.</font></b>
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 23:50, 21 October 2021


FLAG-NLS-DhdR

This is a modified version of the transcriptional repression factor DhdR, which is responsive to the oncometabolite D-2-HG. DhdR is a transcriptional repression factor isolated from the bacteria Achromobacter denitrificans . The sequence was human codon optimized for expression in mammalian cells. Additionally, the sequence had a nuclear localization sequence attached to the end, for transport into the nucleus as part of our genetic reporter system, and a FLAG tag attached to the end.

T--Duke--DhdR gel.png
Figure 1: Gel pic for FLAG-NLS-DhdR (bacterial repression factor) against a 2-log ladder

As part of our goal to construct a biosensor system for the compound D-2-HG, we will utilize a plasmid expressing the DhdR gene to provide baseline expression of this repressor gene. In a wild-type environment, without the presence of DhdR, we expect normal expression of the reporter protein. However, when DhdR is present, it will bind to the DhdO binding site, allosterically blocking the transcription of our reporter gene. When D-2-HG is elevated, particularly in IDH1 mutant cells, it binds to DhdR, releasing it from the binding site. This allows for transcription of the downstream reporter protein sequence, resulting in brighter expression that is visible in our in vivo droplet system. Since D-2-HG levels are elevated due to the IDH1 mutation, we expect that there will be an increase in fluorescence or luminescence due to the release of the allosteric transcription factor caused by the binding of the upregulated oncometabolite. When we perform drug screening assays on our completed co-culture system, we will associate decreased fluorescence or luminescence with lower levels of D-2-HG, which is associated with a variety of downstream metabolic impacts.

A schematic for the operation of our system is shown below. T--Duke--DhdrSchematic.png

Figure 1: This figure outlines the mechanism of the interaction between the DhdR allosteric transcription factor and the dhdO binding site.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 675
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 310
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 186
    Illegal BsaI site found at 303