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Team IGEM Paris 2011

Building and characterizing the YFP concentrator design

This system is an improvement of the original experiment with the GFP. To see the fluorescence better, we decided to concentrate the YFP fluorescent molecules fused to TetR on the TetO array to make them more visible in the cell. We have been kindly given the plasmids containing the YFP:TetR construct and the TetO array by D. Lane. We biobricked the YFP:TetR construct and the TetO array so that they can be used by the synthetic biology community. The results are presented here in detail.

Abstract

Results for the YFP concentrator:

We successfully BioBricked both the YFP:TetR (BBa_K606025) and the TetO Array (BBa_K606026) constructs and sent them to the registry

We integrated our construct both in E.coli and B.subtilis

We characterized the YFP:TetR fusion protein both in E.coli and B.subtilis

We characterized the TetO array in E.coli

We've done E.coli to B.subtilis diffusion experiments (with negative results)

Design overview

YFP:TetR/TetO array system

More information on the design here.

Parts and biobrick system construction

YFP:TetR construction

Cloning plan of YFP:TetR construction

TetO array construction

Cloning plan of TetO array construction

Characterisation of YFP:TetR fusion protein and TetO array

Testing the YFP:tetR and TetO array strains from D. Lane

In the article describing the YFP:TetR fusion protein[1], E. coli strains are growing at 20°C to avoid protein aggregation. But nanotubes between B. subtilis have only been observed at 37°C. We tested different possibilities: at 37°C or 30°C and different concentrations of arabinose (0% - 0,1% -0,2%) to deal with protein aggregation.

YFP:TetR / TetO array : 37°C
YFP:TetR / TetO array induced with no arabinose in E. Coli from Dave Lane plasmids.	YFP:TetR / TetO array induced with 0,2% arabinose in E. Coli from Dave Lane plasmids.

YFP:TetR / TetO array : 30°C
YFP:TetR / TetO array induced with no arabinose in E. Coli from Dave Lane plasmids.	YFP:TetR / TetO array induced with 0,2% arabinose in E. Coli from Dave Lane plasmids.

With both YFP:TetR and TetO array plasmids, we can see few spots of fluorescence with 0,2% arabinose. Because there are in the cell extremity, we can suppose that it is concentrated fluorescence in TetO arrays. Nevertheless the protein aggregation is obvious when there is only YFP:tetR at 30°C and 37°C in E. coli.

More pictures and information on the notebook here.

Characterization: Biobricked TetO Array

Microscopy of double transformed pFX234 / Biobricked TetO Array in E. Coli

To characterize this part properly, we took pictures of different strains containing TetO alone; YFP:TetR; or both TetO and YFP:TetR. In each case we made a control where the promoter was not induced with arabinose in E. coli (double transformated with pFX234 and TetO Array).

Biobricked TetO array only: 37°C
TetO array induced with no arabinose on E. Coli .	TetO array induced with 0,2% arabinose on E. Coli .

YFP:TetR only: 37°C
YFP:TetR induced with no arabinose on E. Coli .	YFP:TetR induced with 0,2% arabinose on E. Coli .

YFP:TetR + Biobricked TetO array: 37°C
YFP:TetR-TetO, full construct induced with no arabinose on E. Coli .	YFP:TetR-TetO, full construct induced with 0,2% arabinose on E. Coli .

YFP:TetR and YFP:TetR + Biobricked TetO array: 37°C - Zoom and comparison
YFP:TetR induced with 0,2% arabinose on E. Coli .	YFP:TetR-TetO, full construct induced with 0,2% arabinose on E. Coli .

The pictures of TetO alone show no YFP fluorescence, which was expected because there is no YFP sequence in these plasmids.
The TetR-YFP construct which (emitter part) occasionally shows gross aggregated YFP.
After observing the cells carrying both the TetO and YFP:TetR constructs, we can obviously distinguish glowing dots in some cells. They reflect the behavior we expected. Indeed, appearance of dots (red arrow) shows that the receiver (TetO array) actually links tightly to the YFP:TetR fusion protein!

Microscopy of ibpA mCherry double transformated in E. Coli

We wanted to be able to distinguish precisely the fusion protein aggregates from the YFP:TetR biding to TetO array. We transformated ibpA mCherry cells (expressing a mcherry in a agregation chaperon protein, courtesy of Anne-Sophie Coquel, Inserm U1001) with pFX234 YFP:TetR and biobricked TetO Array plasmids to differenciate TetO array foci from YFP:TetR aggregates.

Case of YFP:TetR over-expression by arabinose induction

Microscopy shows overlap for most foci and mCherry agregation but some foci do not exhibit red fluorescence.
Hopefully we don't expect to get high concentrations of YFP:TetR in receiver cells so aggregates will not happen in them.

Case of YFP:TetR low expression by arabinose induction

Microscopy shows that most of aggregates are gone and we have more not-overlaping foci.

The ibpA experiment confirms what we suspected: we can easily distinguish aggregates from YFP:TetR biding to TetO spots. This means the experiments with this system can be carried on easily and will give us a clear response.

Characterization: Biobricked YFP:TetR

Microscopy of double transformed pDAG470 / Biobricked YFP:TetR in E. Coli

To characterize this part properly, we took pictures of different strains containing YFP:TetR alone and both TetO array (from pDAG479, D.Lane) and expression YFP:TetR system (BBa_K606027).

Biobricked YFP:TetR only
Biobricked expression YFP:tetR system in E. coli.	Biobricked expression YFP:tetR system in E. coli.

Biobricked YFP:TetR and TetO Array
Biobricked expression YFP:tetR system and TetO Array from D. Lane in E. coli.	Biobricked expression YFP:tetR system and TetO Array from D. Lane in E. coli.

The TetR-YFP construct which (emitter part) occasionally shows gross aggregated YFP.
After observing the cells carrying both the TetO and YFP:TetR constructs, we can obviously distinguish glowing dots in some cells. They reflect the behavior we expected.

Testing the nanotubes with this design

The YFP concentrator This design relies on a TetO-array which allow us to concentrate YFP-TetR fusion proteins. We were able to to a E.coli to B.subtilis diffusion through nanotubes experiment with this design.

References and acknowledgments

Kinetics of plasmid segregation in Escherichia coli, Scott Gordon, Jerôme Rech, David Lane and Andrew Wright, Molecular Biology, available here

YFP concentrator design

Testing nanotubes with the YFP concentrator system

@@ Line 2: / Line 2: @@
 <html>
-<h1>Experiments of the YFP concentrator design</h1>
+<h1>Building and characterizing the YFP concentrator design</h1>
-<p>This system is an improvement of the original experiment with the GFP. In fact, to see the fluorescence better, we decided to concentrate the YFP fluorescent molecules fused to TetR on the TetO array to make them more visible in the cell. We have been kindly given the plasmids containing YFP:TetR construct and TetO array by D. Lane. In order to use this system according to our designs, we biobricked the TetO array and incorporated it in a plasmid compatible with <i>B.subtilis</i> (pHM3). In fact, this plasmid is also compatible with <i>E.coli</i>, which permitted us to successfully characterize it in this strain. The results are presented here in detail.</p>
+<p>This system is an improvement of the original experiment with the GFP. To see the fluorescence better, we decided to concentrate the YFP fluorescent molecules fused to TetR on the TetO array to make them more visible in the cell. We have been kindly given the plasmids containing the YFP:TetR construct and the TetO array by D. Lane. We biobricked the YFP:TetR construct and the TetO array so that they can be used by the synthetic biology community. The results are presented here in detail.</p>
+<h2>Abstract</h2>
+<div style="margin-left:50px; margin-right:50px; padding: 5px; border:2px solid black;"><b><p>Results for the YFP concentrator:
+<ul>
+     	<li>We successfully BioBricked both the YFP:TetR (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K606025">BBa_K606025</a>) and the TetO Array (<a href="http://partsregistry.org/Part:BBa_K606026">BBa_K606026</a>) constructs and sent them to the registry</li>
+        <li>We integrated our construct both in <i>E.coli</i> and <i>B.subtilis</i></li>
+	<li>We characterized the YFP:TetR fusion protein both in <i>E.coli</i> and <i>B.subtilis</i></li>
+	<li>We characterized the TetO array in <i>E.coli</i></li>
+<li>We've done <i>E.coli</i> to <i>B.subtilis</i> diffusion experiments (with negative results)</li>
+</ul></p></b></div>
+<br/>
 <h2>Design overview</h2>
-<center><img src="https://static.igem.org/mediawiki/2011/9/93/YFP_concentration_principle.jpg">
+<center><img src="https://static.igem.org/mediawiki/2011/9/93/YFP_concentration_principle.jpg"></center>
-<p>Cloning of TetO array construction</center></p>
+<center><p>YFP:TetR/TetO array system</p></center>
-<p> More information on the design here : <a href="https://2011.igem.org/Team:Paris_Bettencourt/GFPLac_diffusion ">link</a></p>
+<p>More information on the design <a href="https://2011.igem.org/Team:Paris_Bettencourt/GFPLac_diffusion ">here</a>.</p>
 <h2>Parts and biobrick system construction</h2>
@@ Line 21: / Line 31: @@
 <h3>TetO array construction</h3>
-<center><img src="https://static.igem.org/mediawiki/2011/a/a0/TetOarray2.jpg">
+<center><img src="https://static.igem.org/mediawiki/2011/a/a0/TetOarray2.jpg"></center>
-<p>Cloning plan of TetO array construction</center></p>
+<center><p>Cloning plan of TetO array construction</p></center>
-<h2>Results</h2>
+<h2>Characterisation of YFP:TetR fusion protein and TetO array</h2>
-</html>
-Start with the good news : <br>We succeeded to biobrick the TetO array and the next step is to characterize it. The plan is to do microscopy in ''E. coli'' double transformed with YFP:tetR WT and tetO array (Biobricked), ''E. coli'' with YFP:tetR WT only, ''E. coli'' with tetO array BB only, <i>B.subtilis</i> with tetO array (in pHM3 or K090403). <br><br>
-The bad news : <br>
-We have a lot of trouble to biobrick the YFP:tetR so it will be done if we succeed to go to the World Jamboree.
-<html>
-<h3>Testing the YFP:tetR and tetO array strains from D. Lane</h3>
+<h3>Testing the YFP:tetR and TetO array strains from D. Lane</h3>
-In the article <a href="https://2011.igem.org/Team:Paris_Bettencourt/Experiments/YFP_TetR_diffusion#references">[1]</a>, <i>E. coli</i> strains are growing at 20°C to avoid protein aggregation, but the problem is that nanotubes between <i>B. subtilis</i> have only been shown to exist at 37°C.
+<p>In the article describing the YFP:TetR fusion protein<a href="https://2011.igem.org/Team:Paris_Bettencourt/Experiments/YFP_TetR_diffusion#references">[1]</a>, <i>E. coli</i> strains are growing at 20°C to avoid protein aggregation. But nanotubes between <i>B. subtilis</i> have only been observed at 37°C.
-We tested different possibilities : at 37°C or 30°C and concentration of arabinose (0% - 0,1% -0,2%) to deal with protein aggregation.
+We tested different possibilities: at 37°C or 30°C and different concentrations of arabinose (0% - 0,1% -0,2%) to deal with protein aggregation.</p>
 </html>
 <center>
 {| border="1" class="wikitable" style="text-align: center;" align="center"
-|+tetR:YFP / TetO array : 37°C
+|+YFP:TetR / TetO array : 37°C
 |-
-|[[File:TetR0_YFP02.jpg|350px|thumb|center|tetR:YFP / TetO array inducted with no arabinose on E. Coli from Dave Lane plasmids.]]
+|[[File:TetR0_YFP02.jpg|350px|thumb|center|YFP:TetR / TetO array induced with no arabinose in E. Coli from Dave Lane plasmids.]]
-|[[File:TetR02_YFP03.jpg|350px|thumb|center|tetR:YFP / TetO array inducted with 0,2% arabinose on E. Coli from Dave Lane plasmids.]]
+|[[File:TetR02_YFP03.jpg|350px|thumb|center|YFP:TetR / TetO array induced with 0,2% arabinose in E. Coli from Dave Lane plasmids.]]
 |}
 {| border="1" class="wikitable" style="text-align: center;"
-|+tetR:YFP / TetO array : 30°C
+|+YFP:TetR / TetO array : 30°C
 |-
-|[[File:TetRTetO0_YFP01.jpg|350px|thumb|center|tetR:YFP / TetO array inducted with no arabinose on E. Coli from Dave Lane plasmids.]]
+|[[File:TetRTetO0_YFP01.jpg|350px|thumb|center|YFP:TetR / TetO array induced with no arabinose in E. Coli from Dave Lane plasmids.]]
-|[[File:TetRTetO02_YFP01.jpg|350px|thumb|center|tetR:YFP / TetO array inducted with 0,2% arabinose on E. Coli from Dave Lane plasmids.]]
+|[[File:TetRTetO02_YFP01.jpg|350px|thumb|center|YFP:TetR / TetO array induced with 0,2% arabinose in E. Coli from Dave Lane plasmids.]]
 |}
 </center>
@@ Line 54: / Line 58: @@
 <html>
-<p>With the combination of YFP:TetR and TetO array plasmids, we can see few spots of fluorescence with 0,2% arabinose and because there are in the cell extremity, we can suppose that it is concentrated fluorescence in TetO array. Nevertheless the protein aggregation is very effective when there is only YFP:tetR at 30°C and 37°C in <i>E. coli</i>.</p>
+<p>With both YFP:TetR and TetO array plasmids, we can see few spots of fluorescence with 0,2% arabinose. Because there are in the cell extremity, we can suppose that it is concentrated fluorescence in TetO arrays. Nevertheless the protein aggregation is obvious when there is only YFP:tetR at 30°C and 37°C in <i>E. coli</i>.</p>
-<p>More pictures and information on the notebook here : <a href="https://2011.igem.org/Team:Paris_Liliane_Bettencourt/Notebook/2011/08/03/#Kevin">link</a></p>
+<p>More pictures and information on the notebook <a href="https://2011.igem.org/Team:Paris_Liliane_Bettencourt/Notebook/2011/08/03/#Kevin">here</a>.</p>
-<h3>Characterization: Biobricked TetO Array's running way </h3>
+<h3>Characterization: Biobricked TetO Array</h3>
-<h4>Microscopy of double transformed pFX234 / Biobricked TetO Array <i>E. Coli</i></h4>
+<h4>Microscopy of double transformed pFX234 / Biobricked TetO Array in <i>E. Coli</i></h4>
-</html>
-In order to do this characterization, we took pictures of different plasmids containing only TetO; TetR + YFP; TetO + TetR + YFP. in each case we made a control by non inducing the promoter with arabinose in ''E. coli'' (double transformated with pFX234 and TetO Array).
+<p>To characterize this part properly, we took pictures of different strains containing TetO alone; YFP:TetR; or both TetO and YFP:TetR. In each case we made a control where the promoter was not induced with arabinose in <i>E. coli</i> (double transformated with pFX234 and TetO Array).</p>
+</html>
 <center>
 {| border="1" class="wikitable" style="text-align: center;" align="center"
-|+ tetO array : 37°C
+|+ Biobricked TetO array only: 37°C
 |-
-|[[File:teto_minus_Fluo20.jpg|350px|thumb|center|tetO / TetO array inducted with no arabinose on E. Coli .]]
+|[[File:teto_minus_Fluo20.jpg|350px|thumb|center|TetO array induced with no arabinose on E. Coli .]]
-|[[File:teto_arab_Fluo20.jpg|350px|thumb|center|tetO / TetO array inducted with 0,2% arabinose on E. Coli .]]
+|[[File:teto_arab_Fluo20.jpg|350px|thumb|center|TetO array induced with 0,2% arabinose on E. Coli .]]
 |}
 {| border="1" class="wikitable" style="text-align: center;"
-|+tetR:YFP : 37°C
+|+ YFP:TetR only: 37°C
 |-
-|[[File:yfp_tetr_minus_fluo20.jpg|350px|thumb|center|tetR:YFP / TetR-YFP inducted with no arabinose on E. Coli .]]
+|[[File:yfp_tetr_minus_fluo20.jpg|350px|thumb|center|YFP:TetR induced with no arabinose on E. Coli .]]
-|[[File:yfp_tetr_Arab_fluo20.jpg|350px|thumb|center|tetR:YFP / TetR-YFP inducted with 0,2% arabinose on E. Coli .]]
+|[[File:yfp_tetr_Arab_fluo20.jpg|350px|thumb|center|YFP:TetR induced with 0,2% arabinose on E. Coli .]]
 |}
 {| border="1" class="wikitable" style="text-align: center;"
-|+tetR:YFP / TetO array : 37°C
+|+YFP:TetR + Biobricked TetO array: 37°C
 |-
-|[[File:yfp_tetr_teto_minus_fluo20_2s.jpg|350px|thumb|center|TetR:YFP-tetO/ full construct inducted with no arabinose on E. Coli .]]
+|[[File:yfp_tetr_teto_minus_fluo20_2s.jpg|350px|thumb|center|YFP:TetR-TetO, full construct induced with no arabinose on E. Coli .]]
-|[[File:yfp_tetr_teto_Arab_fluo20_2-1.jpg|350px|thumb|center|TetR:YFP-tetO / full construct inducted with 0,2% arabinose on E. Coli .]]
+|[[File:yfp_tetr_teto_Arab_fluo20_2-1.jpg|350px|thumb|center|YFP:TetR-TetO, full construct induced with 0,2% arabinose on E. Coli .]]
 |}
 {| border="1" class="wikitable" style="text-align: center;"
-|+tetR:YFP and tetR:YFP / TetO array : 37°C - Zoom and comparison
+|+YFP:TetR and YFP:TetR + Biobricked TetO array: 37°C - Zoom and comparison
 |-
-|[[File:yfp_tetr_zoom.jpg|350px|thumb|center|TetR:YFP inducted with 0,2% arabinose on E. Coli .]]
+|[[File:yfp_tetr_zoom.jpg|350px|thumb|center|YFP:TetR induced with 0,2% arabinose on E. Coli .]]
-|[[File:yfp_tetr_teto_zoom.jpg|350px|thumb|center|tetR:YFP-TetO / full construct inducted with 0,2% arabinose on E. Coli .]]
+|[[File:yfp_tetr_teto_zoom.jpg|350px|thumb|center|YFP:TetR-TetO, full construct induced with 0,2% arabinose on E. Coli .]]
 |}
 </center>
-The pictures of TetO alone show no YFP activity, which is normal because there is no YFP sequence in these plasmids.<br>
+The pictures of TetO alone show no YFP fluorescence, which was expected because there is no YFP sequence in these plasmids.<br>
-The TetR-YFP construct which constitutes the transmitter part, occasionally shows gross aggregated YFP.<br>
+The TetR-YFP construct which (emitter part) occasionally shows gross aggregated YFP.<br>
-After observing the cells carrying both the TetO and YFP:TetR constructs, we can obviously distinguish glowing dots in some cells. They reflect the behavior we expected. Indeed, appearance of dots (red arrow) shows that the receiver (TetO array) actually links tightly to TetR-YFP which is the emitted protein! In fact, you can see a lot of glowing dots, each of them being a concentration of fluorescent molecules (we have highlighted some of them with red arrows).
+After observing the cells carrying both the TetO and YFP:TetR constructs, we can obviously distinguish glowing dots in some cells. They reflect the behavior we expected. Indeed, appearance of dots (red arrow) shows that the receiver (TetO array) actually links tightly to the YFP:TetR fusion protein!
 <h4>Microscopy of ibpA mCherry double transformated in <i>E. Coli</i></h4>
-We transformated ibpA mCherry cells (expressing a mcherry in a agregation chaperon protein, with courtesy of Anne-Sophie Coquel, Inserm U1001) with pFX234 YFP:TetR and biobricked TetO Array plasmids to differenciate TetO array foci than agregation.
+We wanted to be able to distinguish precisely the fusion protein aggregates from the YFP:TetR biding to TetO array.
+We transformated ibpA mCherry cells (expressing a mcherry in a agregation chaperon protein, courtesy of Anne-Sophie Coquel, Inserm U1001) with pFX234 YFP:TetR and biobricked TetO Array plasmids to differenciate TetO array foci from YFP:TetR aggregates.
 *Case of YFP:TetR over-expression by arabinose induction
@@ Line 103: / Line 108: @@
 [[File:microscopy_yfp_ibpa.jpg|center|]]
-Microscopy shows overlap for most foci and mCherry agregation but there is some foci that is alone.<br>
+Microscopy shows overlap for most foci and mCherry agregation but some foci do not exhibit red fluorescence.<br>
-Hopefully we don't expect to get high concentration of YFP:tetR in receiver cell so it will be ok.
+Hopefully we don't expect to get high concentrations of YFP:TetR in receiver cells so aggregates will not happen in them.
 *Case of YFP:TetR low expression by arabinose induction
@@ Line 110: / Line 115: @@
 [[File:microscopy_yfp_ibpa2.jpg|center|]]
-Microscopy shows that most of agregation are gone and we have more not-overlaping foci.<br> We could manage to get less agregation if we deal with the arabinose induction.<br>
+Microscopy shows that most of aggregates are gone and we have more not-overlaping foci.<br>
 <html>
-<h2>Future</h2>
+<p>The ibpA experiment confirms what we suspected: <em>we can easily distinguish aggregates from YFP:TetR biding to TetO spots</em>. This means the experiments with this system can be carried on easily and will give us a clear response.</p>
+<h3>Characterization: Biobricked YFP:TetR</h3>
+<h4>Microscopy of double transformed pDAG470 / Biobricked YFP:TetR in <i>E. Coli</i></h4>
+<p>To characterize this part properly, we took pictures of different strains containing YFP:TetR alone and both TetO array (from pDAG479, D.Lane) and expression YFP:TetR system (BBa_K606027).</p>
 </html>
-Next step is to biobrick the YFP:TetR fusion protein so we can finish the cloning plan and put the system in ''B. subtilis''. Hopefully we can improve our GFP diffusion experiments and have a better characterisation of nanotubes !
+<center>
+{| border="1" class="wikitable" style="text-align: center;" align="center"
+|+ Biobricked YFP:TetR only
+|-
+|[[File:yfptetrbb_trans.jpg|350px|thumb|center|Biobricked expression YFP:tetR system in E. coli.]]
+|[[File:yfptetrbb_Fluo2.jpg|350px|thumb|center|Biobricked expression YFP:tetR system in E. coli.]]
+|}
+{| border="1" class="wikitable" style="text-align: center;" align="center"
+|+ Biobricked YFP:TetR and TetO Array
+|-
+|[[File:yfptetrbb_teto_trans.jpg|350px|thumb|center|Biobricked expression YFP:tetR system and TetO Array from D. Lane in E. coli.]]
+|[[File:yfptetrbb_teto__Fluo2.jpg|350px|thumb|center|Biobricked expression YFP:tetR system and TetO Array from D. Lane in E. coli.]]
+|}
+</center>
+<html>
+The TetR-YFP construct which (emitter part) occasionally shows gross aggregated YFP. <br>
+After observing the cells carrying both the TetO and YFP:TetR constructs, we can obviously distinguish glowing dots in some cells. They reflect the behavior we expected.
+<h2>Testing the nanotubes with this design</h2>
+<table>
+<tr>
+  <td style="width:200px; text-align:center"><a href="https://2011.igem.org/Team:Paris_Bettencourt/Experiments/YFP_TetR_diffusion_experiments"><img style="width:150px" src="https://static.igem.org/mediawiki/2011/2/2d/Yfp_diff_button_pb.png"></a>
+  </td>
+  <td><b><a href="https://2011.igem.org/Team:Paris_Bettencourt/Experiments/YFP_TetR_diffusion_experiments">The YFP concentrator</a></b> This design relies on a TetO-array which allow us to concentrate YFP-TetR fusion proteins. We were able to to a <em><i>E.coli</i> to <i>B.subtilis</i> diffusion through nanotubes experiment</em> with this design.
+  </td>
+</tr>
+<tr>
+</table>
 <html>
-</ul>
 <div id="citation_box">
 <p id="references">References and acknowledgments</p>
@@ Line 126: / Line 162: @@
 </div>
 <br>
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@@ Line 170: / Line 205: @@
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-		        <li id="disclai
+		        <li id="disclaimer"><a href="/2011.igem.org:General_disclaimer" title="2011.igem.org:General disclaimer">Disclaimers</a></li>
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+<div id="scroll_right"><a href="https://2011.igem.org/Team:Paris_Bettencourt/Experiments/YFP_TetR_diffusion_experiments"><img src="https://static.igem.org/mediawiki/2011/e/e0/Arrow-right-big.png" style="width:100%;"></a><a href="https://2011.igem.org/Team:Paris_Bettencourt/Experiments/YFP_TetR_diffusion_experiments">Testing nanotubes with the YFP concentrator system</a></div>
+</html>

Team:Paris Bettencourt/Experiments/YFP TetR diffusion