Difference between revisions of "Team:UGent Belgium/Filament"

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<h2> Filament </h2>
 
<h2> Filament </h2>
 
<h3> Overview </h3>
 
<h3> Overview </h3>
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<p> The general idea is to dissolve PLA and biotin in a solvent that allows good dissolvement for both components without destructing or changing their inherent character. After dissolving the PLA and biotin, the PLA (and hopefully the attached/included biotin) must be solidified again. The solidified PLA should be melted and formed into pellets that subsequently can be fed to an extruder to make PLA filament with biotin. The PLA-biotin filament can then be used to print our awesome water collector whereon proteins can be adhered via the well known biotin-(strept)avidin interaction. </p>
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<p> In the filament work package, we try to produce a plastic filament that’s activated and susceptible biological appendage. The filament can subsequently be used for printing the desired 3D structure.
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Poly-lactate (PLA) was chosen as basic plastic carrier because of it’s biodegradable, optimal temperature, and generally easy-to-print characteristics.
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In order to enhance the function of the PLA, cfr. improve the condensation capacity, biological nucleation proteins will need to be fused to it. In order to get the proteins attached to the final structure, either purified or expressed on the membrane of whole cells, a moiety needs to be present in the filament that captures them, binds them en keeps them there (even while rinsing, or in flooded conditions). </p>
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<br>
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<div class="includeimage">
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<p><img src="https://static.igem.org/mediawiki/2016/d/df/T--Ugent_Belgium--filament1.png" alt="fig1" height="266" width="800"></p>
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</div>
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<br>
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      <p><i>General overview; 3D printed structure that's activated with biotin, will be succeptible for adhesion of proteins via biotin-avidin complexation</i></p>
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<br>
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<p>We’ve chosen to assess the biotin-(strept)avidin complex, since it’s a very robust and in most circumstances a fairtin-activated PLly forgiving binding. Also, thanks to the inertial properties of biotin, the adherence or impregnation to the filament can be accomplished in quite harsh environments, without damaging any structures.</p>
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<p>There are three straight-forward approaches to biologically publicizing the biotin on the PLA filament:</p>
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<p>1) Biotin can be actively linked to the filament or final structures</p>
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<p>2) Biotin can be impregnated in PLA from where filament can be made which can then be used for 3D printing</p>
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<p>3) Biotin can be coated on the filament or final structures</p>
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<br>
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<p>Since the first option is generally far to expensive to make bulk quantities of biotin-activated PLA (Salem <i>et al.</i>, 2001; Ben-Shabat <i>et al.</i>, 2006; Weiss <i>et al.</i>, 2007), only option 2 and 3 will be assessed in this work package.</p>
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<h3>  Method 2: Impregnation of biotin </h3>
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<p>In a first method we attempted, we tried to impregnate the biotin in the plastic. Therefore, we looked for a chemical condition in which a vast amount of PLA and biotin could be dissolved without changing or destroying their integrity. This condition was obtained by heating biotin-saturated dimethylformamide (DMF) to 130°C and dissolving PLA in it.</p>
  
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<h3> Dissolving </h3>
 
 
<div class="includeimage">
 
<div class="includeimage">
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<p><img src="https://static.igem.org/mediawiki/2016/c/c7/T--UGent_Belgium--dissolvement_filament.png" alt="Dissolvement" height="170" width="170"></p>
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<p><img src="https://static.igem.org/mediawiki/2016/d/d4/T--Ugent_Belgium--filament2.png" alt="fig2" height="599" width="449"></p>
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      <p>From literature, we found that heated DMF (dimethylformamid) could be a good option for dissolving solid PLA and biotin, without disrupting the polymer or destroying the biotin. Due to the momentarily lack of biotin in the lab, we already went out and test the solubility of PLA in hot DMF and succeeded to dissolve 0,55g PLA in 8ml (68,75 g/l) without reaching its maximum, although the viscosity did increase by the end, by heating DMF in a hot oil bath (80-90°C) with a reflux column for safety (see set-up in picture on the left).</p>
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</div>
 
</div>
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<br>     
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<p><i>Figure 2: Experimental setup for the impregnation of biotin</i></p>
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<p>After thoroughly mixing the dissolved PLA and biotin, the PLA (with biotin) can be crashed out of solution by adding solution to an excess of ethanol, saturated with biotin at room-temperature.</p>
  
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<h3>  Solidifying </h3>
 
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<p>After dissolving the PLA, we wanted to precipitate it. Since DMF is miscible with water and ethanol, and PLA is not, we wanted to alter the polarity of the DMF by mixing it with either of those, and doing so, forcing the PLA out of solution. Since PLA is hygroscopic, we used room temperature ethanol and just added the hot DMF directly to it, resulting in a cotton-like debris (PLA). This was washed again with ethanol and dried in a 70°C oven.</p>
 
  
 
<h3>  Melting </h3>
 
<h3>  Melting </h3>

Revision as of 21:24, 18 October 2016

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Filament

Overview

In the filament work package, we try to produce a plastic filament that’s activated and susceptible biological appendage. The filament can subsequently be used for printing the desired 3D structure. Poly-lactate (PLA) was chosen as basic plastic carrier because of it’s biodegradable, optimal temperature, and generally easy-to-print characteristics. In order to enhance the function of the PLA, cfr. improve the condensation capacity, biological nucleation proteins will need to be fused to it. In order to get the proteins attached to the final structure, either purified or expressed on the membrane of whole cells, a moiety needs to be present in the filament that captures them, binds them en keeps them there (even while rinsing, or in flooded conditions).


fig1


General overview; 3D printed structure that's activated with biotin, will be succeptible for adhesion of proteins via biotin-avidin complexation


We’ve chosen to assess the biotin-(strept)avidin complex, since it’s a very robust and in most circumstances a fairtin-activated PLly forgiving binding. Also, thanks to the inertial properties of biotin, the adherence or impregnation to the filament can be accomplished in quite harsh environments, without damaging any structures.

There are three straight-forward approaches to biologically publicizing the biotin on the PLA filament:

1) Biotin can be actively linked to the filament or final structures

2) Biotin can be impregnated in PLA from where filament can be made which can then be used for 3D printing

3) Biotin can be coated on the filament or final structures


Since the first option is generally far to expensive to make bulk quantities of biotin-activated PLA (Salem et al., 2001; Ben-Shabat et al., 2006; Weiss et al., 2007), only option 2 and 3 will be assessed in this work package.

Method 2: Impregnation of biotin

In a first method we attempted, we tried to impregnate the biotin in the plastic. Therefore, we looked for a chemical condition in which a vast amount of PLA and biotin could be dissolved without changing or destroying their integrity. This condition was obtained by heating biotin-saturated dimethylformamide (DMF) to 130°C and dissolving PLA in it.

fig2


Figure 2: Experimental setup for the impregnation of biotin

After thoroughly mixing the dissolved PLA and biotin, the PLA (with biotin) can be crashed out of solution by adding solution to an excess of ethanol, saturated with biotin at room-temperature.

Melting

Since PLA pellets for extrusion should be solid and not bigger than 4mm³, and not like a ball of cotton, we tried to melt it in a test tube using a Bunsen flame. This resulted in baking, discoloring and thus destroying of the polymer, probably due to too direct and too hot.
Another setup we tested (not with the cotton, since it was already spent, but with normal PLA filament) is a very controlled increase of temperature using a hot oil bath and calmly increasing the temperature up until melting of the PLA without baking. A completely see-through solution was obtained. We cooled it down and obtained a solid pellet which can be reduced in size by simple mechanical force (mixing, cutting...).

Upcoming

  1. When the biotin arrives, redoing the steps with biotin in mixture
  2. Testing biotin availability after solidifying
  3. Making filament when extruder is finished