Team:Bordeaux/Results

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Latest revision as of 02:20, 18 October 2014

Production (click for more info)

We managed to settle the best conditions to produce the biopolymers in maximal amount. Different medium and production time were tested and a special BL21 strain was used.

Purification (click for more info)

The special properties of the ELP were used to purify the produced proteins by inverse thermal cycling (ITC), which is a quick, easy and column-free method for purification of this polymer

Characterization (click for more info)

Characterization was performed according to different physical and chemical methods. As the protein produced is a polymer, it was transformed into a fiber and its resistance and elasticity have been tested.

References

[1] Doreen M. Floss et al. ELASTIN-like polypeptides revolutionize recombinant protein expression and their biomedical application. Trends in Biotechnology Vol.28 No.1 (PMID 19897265)

[2] Dan W. Urry Entropic Elastic Processes in Protein Mechanisms. I. Elastic Structure Due to an Inverse Temperature Transition and Elasticity Due to Internal Chain Dynamics. Journal of Protein Chemistry, Vol. 7, No.. I, 1988

[3] Dan W. Urry. Physical Chemistry of Biological Free Energy Transduction As Demonstrated by Elastic Protein-Based Polymers. J. Phys. Chem. B 1997, 101, 11007-11028

[4] Dan E. Meyer and Ashutosh Chilkoti. Purification of recombinant proteins by fusion with thermally-responsive polypeptides. NATURE BIOTECHNOLOGY VOL 17 NOVEMBER 1999

[5] Trabbic-Carlson et al. (2004), Expression and purification of recombinant proteins from Escherichia coli: Comparison of an elastin-like polypeptide fusion with an oligohistidine fusion. Protein Science, 13: 3274–3284. doi: 10.1110/ps.04931604

[6] K. Trabbic‐Carlson et al. Effect of protein fusion on the transition temperature of an environmentally responsive elastin‐like polypeptide: a role for surface hydrophobicity? Protein Engineering, Design and Selection (2004) 17 (1): 57-66. doi: 10.1093/protein/gzh006

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-<html>
-<p>
-<b>Applications of BBa_K1317003</b><br>
+[[file:Bdx2014 Production.jpg|left|400px]] [https://2014.igem.org/Team:Bordeaux/Results/Production Production (click for more info)]<br><br><br> We managed to settle the best conditions to produce the biopolymers in maximal amount. Different medium and production time were tested and a special BL21 strain was used.<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>
-The protein ELP has been produced in BL21 strain of E.coli. After phase cycling purification it is possible to spin the protein into a wire, thanks to the wet-spinning method. The ELP can have different length regarding the number of copy of the gene. In these experiments, we used 40mer ELP (assemble of two gene copies).<br><br></p>
+----
-<p>
+[[File:Bdx2014 Purification.jpg|left|400px]] [https://2014.igem.org/Team:Bordeaux/Results/Purification Purification (click for more info)]<br><br><br> The special properties of the ELP were used to purify the produced proteins by inverse thermal cycling (ITC), which is a quick, easy and column-free method for purification of this polymer <br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>
-<b>Polymer formation trial:</b><br><br>
+----
+[[File:Bdx2014 Characterization.jpg|left|400px]] [https://2014.igem.org/Team:Bordeaux/Results/Characterization Characterization (click for more info)]<br><br><br> Characterization was performed according to different physical and chemical methods. As the protein produced is a polymer, it was transformed into a fiber and its resistance and elasticity have been tested. <br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>
-In order to obtain polymer wires, we carried out severeal tests by mixing the ELP with different solvants. The following results have been observed:<br><br>
-Sodium Sulfate (Na2So4) at 320 g/L => cottony aspect of the fiber, the structure is not stable<br>
-Diethyl ether (C2H5)2O => No reaction because the drop is trapped. This solvent is not miscible with the polymer
-Acetone (CH3COCH3) => Formation of a white ring<br>
-Heated Sodium Chloride (NaCl 5M) => Formation of a white cloud due to a lack of cohesiveness<br>
-Calcium Chloride (CaCl2 15%) => 1.25% alginate mixture with ELP40 at 10mg/mL were extruded in the calcium chloride solution: a white-colored fiber is obtained.<br>
-This last trial has been performed with 0.75% alginate which seems to be the best experimental condition to get fibers from the ELP<br><br>
-<img class="resultsmounir1" src="https://static.igem.org/mediawiki/parts/2/22/2014-10-07_20_25_40-iGEM_2014_-_Bordeaux.png" alt=""/><br><br>
+===References===
-<center>Fiber obtained by extruding ELP/Alginate in calcium chloride</center><br><br>
-</p>
-<p>
+[1] Doreen M. Floss et al. ''ELASTIN-like polypeptides revolutionize recombinant protein expression and their biomedical application.'' Trends in Biotechnology Vol.28 No.1 (PMID 19897265)
-<b>Phase transition trial: </b><br><br>
+[2] Dan W. Urry ''Entropic Elastic Processes in Protein Mechanisms. I. Elastic Structure Due to an Inverse Temperature Transition and Elasticity Due to Internal Chain Dynamics.'' Journal of Protein Chemistry, Vol. 7, No.. I, 1988
-After getting the ELP40 fiber, it is necessary to wash it with hot water for extraction, the fiber could indeed dissolve at low temperature.<br>
+[3] Dan W. Urry. Physical ''Chemistry of Biological Free Energy Transduction As Demonstrated by Elastic Protein-Based Polymers.'' J. Phys. Chem. B 1997, 101, 11007-11028
-- Control remains transparent<br>
-- The aspect of the fiber changes at ambient temperature (from white to transparent)<br>
-Thanks to the mixture alginate + ELP40 (10 mg/L), we could observe the formation of the polymer and it can be deduced that:<br>
-- In presence of salt, product is losing cohesiveness<br>
-- With contact of hot water, the product is contracting<br><br><br>
-<img class="resultsmounir2" src="https://static.igem.org/mediawiki/parts/e/e4/2014-10-07_21_43_49-Phase_transition.png" alt=""/><br><br>
-<center>transition of the ELP according to temperature</center><br><br>
-</p>
-<p>
-You can see the results on the phase transition on this video: https://www.youtube.com/watch?v=xoTQkoNoZu0<br>
-Wet-spinning and mechanical testing of fibers properties<br>
-The wet-spinning method has been used to create a wire out of the polymer.<br><br>
-<img class="resultsmounir3" src="https://static.igem.org/mediawiki/parts/d/d4/TeamBdx2014_Wet_spin.gif" alt=""/><br><br>
-<center>Overview of the wet-sinning method</center><br><br>
-</p>
-<p>
-A traction machine allows to measure the resistance to rupture of a chosen material, in this case, the fiber obtained by wet-spinning. This experiment consists in placing a little stick of the material to be studied between the jaws of the traction machine. It will pull the stick until its rupture. The lenghtening and the applied force are recorded and then converted into distorsion and pressure data.<br>
-The mechanical testing allow us to state:<br>
-For the alginate<br>
-The experiment done with the alginate is reproducible while using the same conditions, but the fiber ends by breaking.<br>
-Alginate breaks faster because the experiment is carried out at high temperature<br>
-Comparison with carbon nanofibers<br>
-The elasticity is comparable with carbon nanofibers. Thus, ELP has characteristic properties of elasticity due to its polymeric nature.<br>
-The fibers are nevertheless fragile and break easily. Enhancing the resistance should be possible by using another part and fusing the proteins (BBa_K1317001, BBa_K1317002)<br>
-Another trial was carried out with two times more alginate. The result seems to be the same, the fiber breaks fast.<br><br>
-<img class="resultsmounir4" src="https://static.igem.org/mediawiki/parts/f/fe/Bdx2014_Mechanical_traction_1.jpg" alt=""/><br><br>
-<center>The mechanical traction machine</center><br><br>
-</p>
-</html>
+[4] Dan E. Meyer and Ashutosh Chilkoti. ''Purification of recombinant proteins by fusion with thermally-responsive polypeptides.'' NATURE BIOTECHNOLOGY VOL 17 NOVEMBER 1999
+[5] Trabbic-Carlson et al. (2004), ''Expression and purification of recombinant proteins from Escherichia coli: Comparison of an elastin-like polypeptide fusion with an oligohistidine fusion.'' Protein Science, 13: 3274–3284. doi: 10.1110/ps.04931604
+[6] K. Trabbic‐Carlson et al. ''Effect of protein fusion on the transition temperature of an environmentally responsive elastin‐like polypeptide: a role for surface hydrophobicity?'' Protein Engineering, Design and Selection (2004) 17 (1): 57-66. doi: 10.1093/protein/gzh006
 {{:Team:Bordeaux/Pied}}