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− | <p>The proof of SynDrop’s concept consists of several critical steps:</p> | + | <html> |
+ | <h1 class="text-wall-heading">Proof of Concept</h1> | ||
+ | <div class="text-wall-area-box"> | ||
+ | <h2 class="text-wall-area-box-heading">The Composite Proof</h2> | ||
+ | <div class="scroll-area"> | ||
+ | <p class="text-content">We proved that our SynDrop system worked as intended by successfully implementing several critical wet lab and dry lab experiments. First, we have created a model to determine microfluidics variables for optimal liposome synthesis. Second, we synthesized stable biocompatible liposomes and demonstrated an internal transcription and translation of functional proteins. Third, we demonstrated that membrane proteins can successfully integrate into our liposomes. Finally, we constructed working fusion proteins that were able to display a designated tag on the outer membrane. | ||
+ | </p> | ||
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+ | </p> | ||
+ | <button class="read-more-button">Read More</button> | ||
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+ | <div class="pagination-item-wrapper"> | ||
+ | <a class="pagination-anchor"> | ||
+ | <div class="pagination-item"></div> | ||
+ | <span class="pagination-text">Description</span> | ||
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+ | </div> | ||
+ | </div> | ||
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+ | <div class="modal-close"></div> | ||
+ | <div class="modal-content"> | ||
+ | <h1>Proof</h1> | ||
+ | <p>The proof of SynDrop’s concept consists of several critical steps:</p> | ||
<ul> | <ul> | ||
<li>We have specified the parameters for cell-sized liposome (5-30 µm) synthesis by using our COMSOL model.</li> | <li>We have specified the parameters for cell-sized liposome (5-30 µm) synthesis by using our COMSOL model.</li> | ||
</ul> | </ul> | ||
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<p>We have concluded that modifications to microfluidic channels affect our system the most (5 µm change in width increased liposome diameter by almost 6 µm), while viscosity and flow rate regulations proved to be an efficient way to fine-tune liposome size in a range of few micrometers (Fig. 1). Moreover, critical value of flow rate ratio was found (Q = 0.7), at which liposomes stopped forming. | <p>We have concluded that modifications to microfluidic channels affect our system the most (5 µm change in width increased liposome diameter by almost 6 µm), while viscosity and flow rate regulations proved to be an efficient way to fine-tune liposome size in a range of few micrometers (Fig. 1). Moreover, critical value of flow rate ratio was found (Q = 0.7), at which liposomes stopped forming. | ||
</p> | </p> | ||
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<div class="image-container"> | <div class="image-container"> | ||
− | <img src="https://static.igem.org/mediawiki/2018/e/ed/T--Vilnius-Lithuania--dv_fig1a_Model.gif"/> | + | <img src="https://static.igem.org/mediawiki/2018/e/ed/T--Vilnius-Lithuania--dv_fig1a_Model.gif" style="max-width:100%" /> |
<p><strong>Fig. 1 </strong> Visual representation of liposome formation based on baseline experimental parametric values. Phase variables A, B and C here have values of 1, 2 and 3 respectively.</p> | <p><strong>Fig. 1 </strong> Visual representation of liposome formation based on baseline experimental parametric values. Phase variables A, B and C here have values of 1, 2 and 3 respectively.</p> | ||
</div> | </div> | ||
− | < | + | <p>We both stabilized and optimized the production of liposomes by implementing the obtained parameters of microfluidics variables from our COMSOL model calculations. Synthesized liposomes were biocompatible and capable of encapsulating an in vitro transcription-translation system.</p> |
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<p>The core of our project is liposomes - functional, well-characterized minimal systems for membrane protein research. We have successfully implemented a modified OLA (Octanol-assisted liposome assembly) method<sup>1</sup>1 for high throughput production (Fig. 1) of homogenous and cell-sized (Fig. 2), bilayered (Fig. 3) liposomes that are capable of membrane protein synthesis and integration into the membrane (Fig. 4). To prove that synthesis of proteins is viable, we performed GFP protein synthesis inside our liposomes and recorded measurable fluorescence. Additionally, purified GFP was encapsulated to prove high encapsulation efficiency. To validate membrane protein integration into the membrane, pore forming membrane protein a-hemolysin was inserted into liposomes and the leakage of fluorophore molecule calcein was recorded in the outer solution. </p> | <p>The core of our project is liposomes - functional, well-characterized minimal systems for membrane protein research. We have successfully implemented a modified OLA (Octanol-assisted liposome assembly) method<sup>1</sup>1 for high throughput production (Fig. 1) of homogenous and cell-sized (Fig. 2), bilayered (Fig. 3) liposomes that are capable of membrane protein synthesis and integration into the membrane (Fig. 4). To prove that synthesis of proteins is viable, we performed GFP protein synthesis inside our liposomes and recorded measurable fluorescence. Additionally, purified GFP was encapsulated to prove high encapsulation efficiency. To validate membrane protein integration into the membrane, pore forming membrane protein a-hemolysin was inserted into liposomes and the leakage of fluorophore molecule calcein was recorded in the outer solution. </p> | ||
− | <video width="100% | + | |
+ | |||
+ | <div class="image-container"> | ||
+ | <video width="100%" controls> | ||
<source src="https://static.igem.org/mediawiki/2018/f/fc/T--Vilnius-Lithuania--Fig7_Liposomes_formation_video.mp4" type="video/mp4"> | <source src="https://static.igem.org/mediawiki/2018/f/fc/T--Vilnius-Lithuania--Fig7_Liposomes_formation_video.mp4" type="video/mp4"> | ||
</video> | </video> | ||
− | <strong>Fig. 2</strong> High throughput synthesis of liposomes with customly modified OLA method. The video is 30x slowed down. The width of post-junction channel is 200 µm. | + | <p><strong>Fig. 2</strong> High throughput synthesis of liposomes with customly modified OLA method. The video is 30x slowed down. The width of post-junction channel is 200 µm.</p> |
− | + | </div> | |
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− | < | + | <div class="image-container"> |
− | <strong>Fig. | + | <img src="https://static.igem.org/mediawiki/2018/4/4e/T--Vilnius-Lithuania--Fig8_Liposomes.png"> |
+ | <p><strong>Fig. 3</strong> Automatic detection of droplets with SpotCaliper: the droplets are marked with teal colored circles and the diameter of each is measured; b size frequency distribution histogram fitted to Gaussian distribution (teal fit) proves the homogeneity of the liposomes; </p> | ||
+ | </div> | ||
− | < | + | <div class="image-container"> |
+ | <img src="https://static.igem.org/mediawiki/2018/c/c2/T--Vilnius-Lithuania--Fig9_Liposomes.png"> | ||
+ | <p><strong>Fig. 4</strong> Brightfield image of the liposomes that contain IVTT system and plasmid GFP DNA (after incubation); scale bar is 10 µm; b liposomes imaged with FITC: fluorescence confirms that transcription and translation reactions occur inside them; scale bar is 10 µm; c liposomes containing purified GFP protein: all the liposomes exhibit fluorescence validating excellent encapsulation efficiency; scale bar is 20 µm.</p> | ||
+ | </div> | ||
+ | <div class="image-container"> | ||
+ | <img src="https://static.igem.org/mediawiki/2018/3/34/T--Vilnius-Lithuania--Fig10_Liposomes.png"><p> | ||
<Strong>Fig. 5</Strong> <strong>a</strong> concentrated calcein encapsulated within liposomes: the outer solution fluoresces as some of the liposomes inevitably burst releasing calcein into the outside (calcein exhibits fluorescence only after being diluted); <strong>b</strong> box plot comparison of the control (without a-hemolysin) and a group with inserted a-hemolysin; nonparametrical Mann-Whitney U test was used for the statistical evaluation: | <Strong>Fig. 5</Strong> <strong>a</strong> concentrated calcein encapsulated within liposomes: the outer solution fluoresces as some of the liposomes inevitably burst releasing calcein into the outside (calcein exhibits fluorescence only after being diluted); <strong>b</strong> box plot comparison of the control (without a-hemolysin) and a group with inserted a-hemolysin; nonparametrical Mann-Whitney U test was used for the statistical evaluation: | ||
− | the group with a-hemolysin shows statistically significant (p< 0.0001) increase in fluorescence. | + | the group with a-hemolysin shows statistically significant (p< 0.0001) increase in fluorescence.</p> |
+ | </div> | ||
<p><a href="https://2018.igem.org/Team:Vilnius-Lithuania/Design#Liposomes">More about liposome production</a></p> | <p><a href="https://2018.igem.org/Team:Vilnius-Lithuania/Design#Liposomes">More about liposome production</a></p> | ||
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− | < | + | <img src="https://static.igem.org/mediawiki/2018/6/6e/T--Vilnius-Lithuania--Simo_fig6.jpg"> |
− | </ | + | <p> Proteins that possess ß-barrel structures do not fully denature in the presence of SDS if they are unboiled, which leads to unorthodox movement in SDS-PAGE. As BamA only forms ß-barrels when correctly folded, we can evaluate the amount of protein correctly folded and incorporated into the liposome membranes by the amount of unusual product in an SDS-PAGE. |
+ | </p> | ||
+ | </div> | ||
<p><a href="https://2018.igem.org/Team:Vilnius-Lithuania/Design#BAM%20complex">More about Bam complex proteins</a> | <p><a href="https://2018.igem.org/Team:Vilnius-Lithuania/Design#BAM%20complex">More about Bam complex proteins</a> | ||
</p> | </p> | ||
<ul> | <ul> | ||
− | <li>Finally, as our ultimate goal was to use liposomes as a platform for membrane protein research, we constructed membrane proteins that were capable of displaying protein particles on the surface of E. coli, hence showed no warning signs of not being to do the same in liposomes.</li | + | <li>Finally, as our ultimate goal was to use liposomes as a platform for membrane protein research, we constructed membrane proteins that were capable of displaying protein particles on the surface of E. coli, hence showed no warning signs of not being to do the same in liposomes.</li> |
</ul> | </ul> | ||
<p>We successfully constructed multiple novel membrane proteins based on ß-barrel bearing translocators that are capable of displaying protein particles on the surface of E. coli (Fig. 6). As liposomes feature the same lipids as E. coli membranes, and during our project were successfully incorporated with Bam complex proteins, it appears that these proteins will be able to expose fused protein particles as, if not more, efficiently as in bacteria, laying the foundation to reach new molecular evolution horizons. | <p>We successfully constructed multiple novel membrane proteins based on ß-barrel bearing translocators that are capable of displaying protein particles on the surface of E. coli (Fig. 6). As liposomes feature the same lipids as E. coli membranes, and during our project were successfully incorporated with Bam complex proteins, it appears that these proteins will be able to expose fused protein particles as, if not more, efficiently as in bacteria, laying the foundation to reach new molecular evolution horizons. | ||
</p> | </p> | ||
− | < | + | <div class="image-container"> |
− | <strong>Fig. 7</strong> Plate reader results of the absorption at 450 nm wavelength of the bacteria coding for different exposition proteins. Absorption’s intensity is proportional to the efficiency of display. | + | <img src="https://static.igem.org/mediawiki/2018/4/47/T--Vilnius-Lithuania--Simui_bac_expo.jpg"> |
+ | <p><strong>Fig. 7</strong> Plate reader results of the absorption at 450 nm wavelength of the bacteria coding for different exposition proteins. Absorption’s intensity is proportional to the efficiency of display.</p> | ||
+ | </div> | ||
<p><a href="https://2018.igem.org/Team:Vilnius-Lithuania/Design#Surface_display_system">More about surface display</a> | <p><a href="https://2018.igem.org/Team:Vilnius-Lithuania/Design#Surface_display_system">More about surface display</a> | ||
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Latest revision as of 21:28, 4 November 2018
Proof of Concept
The Composite Proof
We proved that our SynDrop system worked as intended by successfully implementing several critical wet lab and dry lab experiments. First, we have created a model to determine microfluidics variables for optimal liposome synthesis. Second, we synthesized stable biocompatible liposomes and demonstrated an internal transcription and translation of functional proteins. Third, we demonstrated that membrane proteins can successfully integrate into our liposomes. Finally, we constructed working fusion proteins that were able to display a designated tag on the outer membrane.
</html>