Difference between revisions of "Team:Vilnius-Lithuania/Design"

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                             Final and critical step in preparing the chip is the selective coating of post-junction channels with PVA (<var>Polyvinyl alcohol</var>) to render them hydrophilic. It is required to prevent the lipid/octanol solution from wetting the inherently hydrophobic inner channel surface of the device. Without this additional coating, liposomes are unable to form. To coat only a part of microfluidic channels is a challenge: PVA must only remain in post-junction channels without any leakage to the other side. To counter the spreading of PVA to pre-junction channels, air is introduced from separate inlets. A correct interphase between air and PVA must form and stay stable for at least several minutes to let the PVA molecules adhere to the surface (Fig. 5). Any PVA contamination to the opposite side makes the whole device unusable. Not only is this process time consuming (at least 15-20 minutes for each microfluidic device) and requires constant supervision, the procedure quite often fails due to human error while controlling the infusion rates of air and PVA.
 
                             Final and critical step in preparing the chip is the selective coating of post-junction channels with PVA (<var>Polyvinyl alcohol</var>) to render them hydrophilic. It is required to prevent the lipid/octanol solution from wetting the inherently hydrophobic inner channel surface of the device. Without this additional coating, liposomes are unable to form. To coat only a part of microfluidic channels is a challenge: PVA must only remain in post-junction channels without any leakage to the other side. To counter the spreading of PVA to pre-junction channels, air is introduced from separate inlets. A correct interphase between air and PVA must form and stay stable for at least several minutes to let the PVA molecules adhere to the surface (Fig. 5). Any PVA contamination to the opposite side makes the whole device unusable. Not only is this process time consuming (at least 15-20 minutes for each microfluidic device) and requires constant supervision, the procedure quite often fails due to human error while controlling the infusion rates of air and PVA.
 
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                          <strong>Fig. 5</strong> A schematic representation of the interphase of air and PVA at the star shaped junction of LipoDrop.
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                            <img src="https://static.igem.org/mediawiki/2018/2/29/T--Vilnius-Lithuania--Fig5_Liposomes.png"/>
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                            <strong>Fig 5 </strong> A schematic representation of the interphase of air and PVA at the star shaped junction of LipoDrop.
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                             After successfully coating the chips, the devices are finally ready for liposome synthesis. An experiment is conducted utilizing an octanol-assisted liposome assembly (OLA) method. Liposomes are formed when three unique phases (solutions - OA phase, LO phase and IA phase) form a correct interphase at the junction (Fig. 6). The <strong>IA</strong> phase (inner aqueous) occupies the inner part of the liposome and contains an IVTT transcription/translation system, DNA, membrane protein integration machinery, chaperones and salts needed for protein synthesis and integration into the membrane of the liposome. The <strong>LO</strong> phase contains lipids (i.e. DOPC, Cholesterol) and organic solvent (i.e. 2-octanol); it can also contain fluorescent lipids, such as Rh PE, for imaging. This phase introduces all of the components that will form the liposomes’ membrane. Octanol acts as an organic solvent for phospholipids. In the OLA method, initially double emulsions are formed; the excess octanol and lipids dewet and separate from the droplet leaving double-layered liposomes.  Octanol removal from liposomes is crucial as the correct bilayer cannot form in excess organic solvent.  Lastly, the <strong>OA</strong> phase (outer aqueous) contains surfactants that help stabilize the droplets at the initial formation and propagation through the microfluidic channels.  
 
                             After successfully coating the chips, the devices are finally ready for liposome synthesis. An experiment is conducted utilizing an octanol-assisted liposome assembly (OLA) method. Liposomes are formed when three unique phases (solutions - OA phase, LO phase and IA phase) form a correct interphase at the junction (Fig. 6). The <strong>IA</strong> phase (inner aqueous) occupies the inner part of the liposome and contains an IVTT transcription/translation system, DNA, membrane protein integration machinery, chaperones and salts needed for protein synthesis and integration into the membrane of the liposome. The <strong>LO</strong> phase contains lipids (i.e. DOPC, Cholesterol) and organic solvent (i.e. 2-octanol); it can also contain fluorescent lipids, such as Rh PE, for imaging. This phase introduces all of the components that will form the liposomes’ membrane. Octanol acts as an organic solvent for phospholipids. In the OLA method, initially double emulsions are formed; the excess octanol and lipids dewet and separate from the droplet leaving double-layered liposomes.  Octanol removal from liposomes is crucial as the correct bilayer cannot form in excess organic solvent.  Lastly, the <strong>OA</strong> phase (outer aqueous) contains surfactants that help stabilize the droplets at the initial formation and propagation through the microfluidic channels.  
 
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                     <div class="image-container">
                             <strong>Fig. 6</strong> A close-up of the phase interface during liposome synthesis; <strong>IA</strong> phase contains elements required for the synthesis  
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                            <img src="https://static.igem.org/mediawiki/2018/7/77/T--Vilnius-Lithuania--Fig6_Liposomes.png"/>
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                             <strong>Fig 6 </strong> A close-up of the phase interface during liposome synthesis; <strong>IA</strong> phase contains elements required for the synthesis  
 
                             and integration of membrane proteins; <strong>LO</strong> phase consists of octanol and lipids that form a lipid bilayer; OA solution  
 
                             and integration of membrane proteins; <strong>LO</strong> phase consists of octanol and lipids that form a lipid bilayer; OA solution  
 
                             carries surfactants that stabilize the initial formation and propagation of the droplets along the microfluidic device.                             
 
                             carries surfactants that stabilize the initial formation and propagation of the droplets along the microfluidic device.                             
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Revision as of 00:17, 18 October 2018

Design and Results

Results

Cell-free, synthetic biology systems open new horizons in engineering biomolecular systems which feature complex, cell-like behaviors in the absence of living entities. Having no superior genetic control, user-controllable mechanisms to regulate gene expression are necessary to successfully operate these systems. We have created a small collection of synthetic RNA thermometers that enable temperature-dependent translation of membrane proteins, work well in cells and display great potential to be transferred to any in vitro protein synthesis system.

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