Team:EPF-Lausanne/R-BphP

From 2009.igem.org

(Difference between revisions)
 
(6 intermediate revisions not shown)
Line 1: Line 1:
{{EPF-Lausanne09}}
{{EPF-Lausanne09}}
 +
<div CLASS="epfltrick">__TOC__
<div CLASS="epfltrick">__TOC__
</div><div CLASS="epfl09">
</div><div CLASS="epfl09">
 +
<html><center>
<html><center>
-
<font size="6" color="#007CBC"><i>R-BphP Project</i></font>  
+
<font size="6" color="#007CBC"><i>R-BphP project </i></font>  
</center></html>
</center></html>
<br>
<br>
Line 11: Line 13:
<br>
<br>
-
== The BphP / PpsR system ==
+
This part of the project was cancelled, due to lack of time. You can read what our idea was on [https://2009.igem.org/Team:EPF-Lausanne/Brainstorming the brainstorming session]
-
 
+
-
PpsR1 is a redox sensitive activator. It binds DNA under anaerobic conditions, and forms a tetramer via a disulfide bond. This interaction is ablated in the mutant PpsR1-C429S; meaning that we should be able to mimic an anaerobic system even under aerobic conditions.
+
-
 
+
-
PpsR2 is a transcriptional repressor, regulated by BphP.
+
-
 
+
-
PpsR1 and PpsR2 bind to TGTN_12ACA possibly arranged in tandem with a 7 base spacer. The affinity for both PpsRs are around 100nM.
+
-
 
+
-
BphP (or BrBphP for the Bradyrhizobium variant) is sensitive to far-red light (~770nm) and controls PpsR1.
+
-
 
+
-
For more info, see [http://www.jbc.org/cgi/content/full/279/43/44407].
+
-
 
+
-
Similar constituents could possibly be derived from R. Palustris as well with the following model derived from [http://www3.interscience.wiley.com/cgi-bin/fulltext/118512333/HTMLSTART]:
+
-
[[Image:R._palustris_circuit.gif]]
+
-
 
+
-
==To find==
+
-
- Define genetic circuit <big>'''Tú'''</big>
+
-
 
+
-
- Look for exact wavelength for the corresponding light receptor<big><span style="color:Blue"> Basile</span></big>
+
-
 
+
-
- Sequences of the genes involved in the pathway (minimal genes to  => [http://www.ncbi.nlm.nih.gov/ Pubmed])
+
-
 
+
-
[[Media:‎ ORS278_BrBphP_genomic_seq.txt|ORS278 BrBphP genomic sequence]]
+
-
 
+
-
[[Media:‎ ORS278_PpsR1_genomic_seq.txt|ORS278 PpsR1 genomic sequence]]
+
-
 
+
-
[[Media:‎ ORS278_PpsR2_genomic_seq.txt|ORS278 PpsR2 genomic sequence]]
+
-
 
+
-
[[Media:‎ ORS278_PpsR1-C429S_protein_seq.txt|ORS278 PpsR2-C429S protein sequence]]
+
-
 
+
-
[[Media: hmuO_ORS278 Brad.txt|ORS278 hmuO genomic sequence]]
+
-
 
+
-
[[Media: ppsR1_prot_RPalustris.txt| R.Palustris CGA009 ppsR1 protein sequence]]
+
-
 
+
-
[[Media: ppsR1_RPalustris.txt| R.Palustris CGA009 ppsR1 genomic sequence]]
+
-
 
+
-
[[Media: ppsR2_prot_RPalustris.txt| R.Palustris CGA009 ppsR2 protein sequence]]
+
-
 
+
-
[[Media: ppsR2_RPalustris.txt| R.Palustris CGA009 ppsR2 genomic sequence]]
+
-
 
+
-
[[Media: CAE001 bphP1.txt| R.Palustris CAE001 bphP1 genomic sequence]]
+
-
 
+
-
[[Media: CGA009 hmuO.txt| R.Palustris CGA009 hmuO genomic sequence]]
+
-
 
+
-
[[Image: bphp_organisation.gif|500px|thumb|center| Organization of the genes downstream the puf operon in Bradyrhizobium and Rps. palustris. BphP: bacteriophytochrome gene; hmuO: heme oxygenase gene.]]
+
-
 
+
-
==Implementation==
+
-
 
+
-
There are two modes in which we can use the system. The minimal complement requires PpsR2 and its regulator BphP. We can generate a hybrid system that uses a well known activator or transcription or a constitutively active promoter which will be repressed via PpsR2 upon exposure to far-red light (770nm).
+
-
 
+
-
It is also possible to reconstitute the entire system including PpsR1-C429S, which will serve as the activator and will be de-coupled from the oxidative state of the cell due to the ablation of the disulfide bond formation.  
+
-
 
+
-
==To do==
+
-
* contact Eric Giraud (giraud@mpl.ird.fr) and ask if he could send us the following material:
+
-
** Bradyrhizobium ORS278
+
-
** The following plasmids:
+
-
*** pBAD::ppsR1
+
-
*** pBAD::ppsR2
+
-
*** pGEM-T::ppsR2/BrbphP
+
-
** the PpsR1-C429S construct
+
-
 
+
-
==Found==
+
-
- Bacterium: Bradyrhizobium  can be ordered on [http://www.lgcstandards-atcc.org/LGCAdvancedCatalogueSearch/ProductDescription/tabid/1068/Default.aspx?ATCCNum=10244&Template=bacteria ATCC]
+
-
 
+
-
 
+
-
 
+
-
<html>
+
-
<p align="center" class="style1"><a href="#top"><img src="https://static.igem.org/mediawiki/2009/thumb/0/06/Up_arrow.png/50px-Up_arrow.png" alt="Back to top" border="0"></a><br></p>
+
-
</html>
+
-
<br>
+
</div><div CLASS="epfl09bouchon"></div>
</div><div CLASS="epfl09bouchon"></div>

Latest revision as of 08:07, 11 October 2009




R-BphP project



This part of the project was cancelled, due to lack of time. You can read what our idea was on the brainstorming session

Retrieved from "http://2009.igem.org/Team:EPF-Lausanne/R-BphP"