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

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                                       <img src="https://static.igem.org/mediawiki/2018/c/cc/T--Vilnius-Lithuania--Fig_1_NEW_su_uzrasu_Liposomes.png"/>
 
                                       <img src="https://static.igem.org/mediawiki/2018/c/cc/T--Vilnius-Lithuania--Fig_1_NEW_su_uzrasu_Liposomes.png"/>
 
<p><strong>Fig 1</strong> The composition of a liposome with encapsulated machinery for membrane protein integration. Size, membrane composition and interior composition can be easily varied.</p>
 
<p><strong>Fig 1</strong> The composition of a liposome with encapsulated machinery for membrane protein integration. Size, membrane composition and interior composition can be easily varied.</p>
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                                  </p>
 
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                     </div>
 
                      
 
                      
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                     <div class="image-container">
 
                     <div class="image-container">
 
                                       <img src="https://static.igem.org/mediawiki/2018/0/0e/T--Vilnius-Lithuania--Fig2_Liposomes.png"/>
 
                                       <img src="https://static.igem.org/mediawiki/2018/0/0e/T--Vilnius-Lithuania--Fig2_Liposomes.png"/>
                                       <strong>Fig 2 a</strong>AutoCAD design for the photomask. There are 16 individual microchannel devices on a
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                                       <p><strong>Fig 2 a</strong>AutoCAD design for the photomask. There are 16 individual microchannel devices on a
                                       single chip. <strong>b</strong> One device consists of three inlets, an outlet and a star-shaped junction.
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                                       single chip. <strong>b</strong> One device consists of three inlets, an outlet and a star-shaped junction.</p>
 
                                   </p>
 
                                   </p>
 
                     </div>
 
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                             <div class="image-container">
 
                             <div class="image-container">
 
                                     <img src="https://static.igem.org/mediawiki/2018/7/7d/T--Vilnius-Lithuania--Fig3_Liposomes.png"/>
 
                                     <img src="https://static.igem.org/mediawiki/2018/7/7d/T--Vilnius-Lithuania--Fig3_Liposomes.png"/>
                                     <strong>Fig 3 </strong> Simplified scheme for microfluidic device preparation. <strong>a-b</strong> the silicon wafer is cleaned and spin-coated with photoresist; <strong>c</strong> the photomask is aligned on the sample and exposed to UV light. <strong>d</strong> sample is submerged to a developer – only the sections that were exposed to the UV light remain intact  on the wafer; <strong>e</strong> PDMS is poured onto the master to create a PDMS mold and left for a bake in the oven; <strong>f</strong> the mold is then separated and prepared further by cleaning and punching inlets and outlets; <strong>e-f</strong> a microscopic slide is prepared by applying a thin layer of PDMS on top; <strong>i</strong> PDMS mold and PDMS covered microscopic slide are plasma treated and connected to each other to produce a final microfluidic chip.
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                                     <p><strong>Fig 3 </strong> Simplified scheme for microfluidic device preparation. <strong>a-b</strong> the silicon wafer is cleaned and spin-coated with photoresist; <strong>c</strong> the photomask is aligned on the sample and exposed to UV light. <strong>d</strong> sample is submerged to a developer – only the sections that were exposed to the UV light remain intact  on the wafer; <strong>e</strong> PDMS is poured onto the master to create a PDMS mold and left for a bake in the oven; <strong>f</strong> the mold is then separated and prepared further by cleaning and punching inlets and outlets; <strong>e-f</strong> a microscopic slide is prepared by applying a thin layer of PDMS on top; <strong>i</strong> PDMS mold and PDMS covered microscopic slide are plasma treated and connected to each other to produce a final microfluidic chip.</p>
 
                                 </p>
 
                                 </p>
 
                   </div>
 
                   </div>
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                         <div class="image-container">
 
                         <div class="image-container">
 
                                 <img src="https://static.igem.org/mediawiki/2018/7/75/T--Vilnius-Lithuania--Fig4_Liposomes.png"/>
 
                                 <img src="https://static.igem.org/mediawiki/2018/7/75/T--Vilnius-Lithuania--Fig4_Liposomes.png"/>
                                 <strong>Fig 4 </strong> Final form of Lipodrop.
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                                 <p><strong>Fig 4 </strong> Final form of Lipodrop.</p>
 
                             </p>
 
                             </p>
 
               </div>
 
               </div>
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                     <div class="image-container">
 
                     <div class="image-container">
 
                             <img src="https://static.igem.org/mediawiki/2018/2/29/T--Vilnius-Lithuania--Fig5_Liposomes.png"/>
 
                             <img src="https://static.igem.org/mediawiki/2018/2/29/T--Vilnius-Lithuania--Fig5_Liposomes.png"/>
                             <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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                             <p><strong>Fig 5 </strong> A schematic representation of the interphase of air and PVA at the star shaped junction of LipoDrop.</p>
 
                         </p>
 
                         </p>
 
           </div>
 
           </div>
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                     <div class="image-container">
 
                     <div class="image-container">
 
                             <img src="https://static.igem.org/mediawiki/2018/7/77/T--Vilnius-Lithuania--Fig6_Liposomes.png"/>
 
                             <img src="https://static.igem.org/mediawiki/2018/7/77/T--Vilnius-Lithuania--Fig6_Liposomes.png"/>
                             <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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                             <p><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.                             
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                             carries surfactants that stabilize the initial formation and propagation of the droplets along the microfluidic device.</p>                            
 
                     </p>
 
                     </p>
 
                 </div>                           
 
                 </div>                           
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                     <div class="image-container">
 
                     <div class="image-container">
 
                             <img src="https://static.igem.org/mediawiki/2018/f/fc/T--Vilnius-Lithuania--Fig7_Liposomes_formation_video.mp4"/>
 
                             <img src="https://static.igem.org/mediawiki/2018/f/fc/T--Vilnius-Lithuania--Fig7_Liposomes_formation_video.mp4"/>
                             <strong>Fig 7 </strong> High throughput formation of cell-sized liposomes. The video is 60x slowed down     
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                             <p><strong>Fig 7 </strong> High throughput formation of cell-sized liposomes. The video is 60x slowed down </p>      
 
                     </p>
 
                     </p>
 
                 </div>
 
                 </div>
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                     <div class="image-container">
 
                     <div class="image-container">
 
                             <img src="https://static.igem.org/mediawiki/2018/4/4e/T--Vilnius-Lithuania--Fig8_Liposomes.png"/>
 
                             <img src="https://static.igem.org/mediawiki/2018/4/4e/T--Vilnius-Lithuania--Fig8_Liposomes.png"/>
                             <strong>Fig 8 </strong> An automatic detection of droplets with SpotCaliper: the droplets are marked with teal colored circles and the  
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                             <p><strong>Fig 8 </strong> An automatic detection of droplets with SpotCaliper: the droplets are marked with teal colored circles and the  
 
                             diameter of each is measured; <strong>b</strong> size frequency distribution histogram fitted to Gaussian distribution (teal fit) proves  
 
                             diameter of each is measured; <strong>b</strong> size frequency distribution histogram fitted to Gaussian distribution (teal fit) proves  
                             the homogeneity of the liposomes; μ=11.853 >µm±0.017 >µm ; SD=0.442 µm ±0.017 µm.                                    
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                             the homogeneity of the liposomes; μ=11.853 >µm±0.017 >µm ; SD=0.442 µm ±0.017 µm. <p></p>                                   
 
                     </p>
 
                     </p>
 
                 </div>                               
 
                 </div>                               
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                             scale bar is 10 µm; <strong>b</strong> liposomes imaged with FITC: fluorescence confirms that transcription and translation
 
                             scale bar is 10 µm; <strong>b</strong> liposomes imaged with FITC: fluorescence confirms that transcription and translation
 
                             reactions occur inside them; scale bar is 10 µm; <strong>c</strong> liposomes containing purified GFP protein: all the
 
                             reactions occur inside them; scale bar is 10 µm; <strong>c</strong> liposomes containing purified GFP protein: all the
                             liposomes exhibit fluorescence validating excellent encapsulation efficiency; scale bar is 20 µm.                                    
+
                             liposomes exhibit fluorescence validating excellent encapsulation efficiency; scale bar is 20 µm.</p>                                   
 
                     </p>
 
                     </p>
 
                 </div>   
 
                 </div>   
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                         <div class="image-container">
 
                         <div class="image-container">
 
                                 <img src="https://static.igem.org/mediawiki/2018/3/34/T--Vilnius-Lithuania--Fig10_Liposomes.png"/>
 
                                 <img src="https://static.igem.org/mediawiki/2018/3/34/T--Vilnius-Lithuania--Fig10_Liposomes.png"/>
                                 <strong>Fig 10 a</strong> concentrated calcein encapsulated within liposomes: the outer solution fluoresces as some of the liposomes
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                                 <p><strong>Fig 10 a</strong> concentrated calcein encapsulated within liposomes: the outer solution fluoresces as some of the liposomes
 
                                 inevitably burst releasing calcein into the outside; <strong>b</strong> box plot comparison of the control (without α-hemolysin) and
 
                                 inevitably burst releasing calcein into the outside; <strong>b</strong> box plot comparison of the control (without α-hemolysin) and
 
                                 a group with inserted α-hemolysin; nonparametrical Mann-Whitney U test was used for the statistical evaluation:
 
                                 a group with inserted α-hemolysin; nonparametrical Mann-Whitney U test was used for the statistical evaluation:
                                 the group with α-hemolysin shows statistically significant (p < 0.0001) increase in fluorescence                                                   
+
                                 the group with α-hemolysin shows statistically significant (p < 0.0001) increase in fluorescence</p>                                                    
 
                         </p>
 
                         </p>
 
                     </div>   
 
                     </div>   

Revision as of 00:57, 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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