Coding

Part:BBa_K1317003

Designed by: Mounir Benkoulouche   Group: iGEM14_Bordeaux   (2014-10-07)

CDS for Elastin like polypeptide (ELP)

This part is made with a consensus of the natural elastin sequence. Elastin is a protein involved in maintaining shape and elasticity of several tissues like skin. It has a high elasticity and resilience as a polymer.

In our project, the consensus sequence allows the use of a minimal pattern of the protein while keeping these particular properties. In an iGEM spirit the consensus itself can be used as a brick to vary length and nature of the polymers with our other biobricks (BBa_K1317001, BBa_K1317002).

The basic length of a polymer is 20 monomers. The coding sequence can be repeated easily using our improvement of the biobrick assembly system to get rid of the Stop Codon. After variation of the length different properties are attributed to the part. For example it can be used as a fusion tag to purify easily any fuse protein thanks to the thermal cycling purification.

ELP indeed have this particularity to solubilize at lower temperature. If it is used in a protein mixture (a lysate for example) after a few cycles of thermal cycling (from 4°C to 50°C), the pure fuse protein is obtained.

This part can be used as a purification tag for easy, quick and column-free purification or as coding sequence for a polymer usable to get fiber with good elasticity and resilience. The polymer is biocompatible, biodegradable and could be used as a tool for surgery or other health applications.

Link on our wiki: [http://2014.igem.org/Team:Bordeaux/Parts/BBa_K1317002 http://2014.igem.org/Team:Bordeaux/Parts/BBa_K1317003]


Usage and Biology

Production and purification of the polymer for downstream applications

Bdx2014 ELP04 01.png

Strain

The protein ELP has been produced in BLR strain of E.coli (F–ompT hsdSB(rB– mB–) gal dcm Δ(srl-recA)306::Tn10)

BLR is more suited for the production of ELP, it is a BL21 derivative in which plasmids containing repetitive sequences are stabilized.

Culture

Culture was performed in LB-glucose (ELP60) or LB-glycerol (ELP20, 40). After inoculation from a preculture the transformed BLR strain was incubated at 37°C in 1L of appropriate LB medium. Induction was performed when the OD reached 0.8 with 0.8mM IPTG (typically after 2.75h for VPGXG20, 5.25h for VPGXG40 and 1.25h for VPGXG60). Temperature was lowered at 25°C. Culture is stopped 21h after induction.

Bdx2014 DO VPGXG20 b.png
Bdx2014 DO VPGXG40.png

Bdx2014 DO VPGXG60.png

Purification by ITC

Cells are lysed and the protein mixture is collected. The ELP is purified by ITC (inverse transition cycling)

Bdx2014 ELP Tt.png
Bdx2014 ITC method.png


Method overview:

- 2ml of polyethylenimine is added to the lysate, the mixture is centrifuged at 14,000rpm for 15min at 4°C.

- The supernatant is saved and brought to room temperature. NaCl (1 to 3M) is added while homogenising the solution.

- The mixture is then centrifuged at 14,000 rpm for 15min at 37°C.

- Supernatent is discarded and pellet is resuspended with 2ml of cold PBS.

- First and second steps are repeated but NaCl 5M is added so the ELP precipitate.

- Third and fourth steps are repeated.

- A final centrifugation is performed at 4°C for 10min (14,000rpm)

- The solution is dialysed and the samples are lyophilized.


Investigating the properties of the polymer

Bdx2014 ELP01.png Bdx2014 ELP02.png Bdx2014 ELP03 01.png


Polymer formation trial:

Fiber obtained by extruding ELP/Alginate in calcium chloride

In order to obtain polymer wires, we carried out severeal tests by mixing the ELP with different solvants. The following results have been observed:

  • Sodium Sulfate (Na2So4) at 320 g/L => cottony aspect of the fiber, the structure is not stable
  • 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
  • Heated Sodium Chloride (NaCl 5M) => Formation of a white cloud due to a lack of cohesiveness
  • 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.

This last trial has been performed with 0.75% alginate which seems to be the best experimental condition to get fibers from the ELP


Phase transition trial:

Phase transition of the ELP according to temperature

After getting the ELP40 fiber, it is necessary to wash it with hot water for extraction, the fiber could indeed dissolve at low temperature.

- Control remains transparent

- The aspect of the fiber changes at ambient temperature (from white to transparent)

Thanks to the mixture alginate + ELP40 (10 mg/L), we could observe the formation of the polymer and it can be deduced that:

- In presence of salt, product is losing cohesiveness

- With contact of hot water, the product is contracting

You can see the results on the phase transition on this video: https://www.youtube.com/watch?v=xoTQkoNoZu0

Wet-spinning and mechanical testing of fibers properties

The wet-spinning method has been used to create a wire out of the polymer.

TeamBdx2014 Wet spin.gif
Overview of the wet-sinning method


The mechanical traction machine

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.

The mechanical testing allow us to state:

  1. For the alginate
    • The experiment done with the alginate is reproducible while using the same conditions, but the fiber ends by breaking.
    • Alginate breaks faster because the experiment is carried out at high temperature
  2. Comparison with carbon nanofibers
    • The elasticity is comparable with carbon nanofibers. Thus, ELP has characteristic properties of elasticity due to its polymeric nature.
    • 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)

Another trial was carried out with two times more alginate. The result seems to be the same, the fiber breaks fast.

All data are available on the wiki page of this part:

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 312
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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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Categories
//chassis/prokaryote/ecoli
Parameters
chassisE.coli (BLR)