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

 
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<h1 class="text-wall-heading">Proof of Concept</h1>
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    <h2 class="text-wall-area-box-heading">The Composite Proof</h2>
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        <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.
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<h1>Demonstrate</h1>
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        <button class="read-more-button">Read More</button>
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<h3>Gold Medal Criterion #4</h3>
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            <span class="pagination-text">Description</span>
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        <h1>Proof</h1>
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        <p>The proof of SynDrop’s concept consists of several critical steps:</p>
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<ul>
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    <li>We have specified the parameters for cell-sized liposome (5-30 µm) synthesis by using our COMSOL model.</li>
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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.
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Teams that can show their system working under real world conditions are usually good at impressing the judges in iGEM. To achieve gold medal criterion #4, convince the judges that your project works. There are many ways in which your project working could be demonstrated, so there is more than one way to meet this requirement. This gold medal criterion was introduced in 2016, so check our what 2016 teams did to achieve their gold medals!
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Please see the <a href="https://2018.igem.org/Judging/Medals">2018 Medals Page</a> for more information.
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                          <img  src="https://static.igem.org/mediawiki/2018/e/ed/T--Vilnius-Lithuania--dv_fig1a_Model.gif" style="max-width:100%" />
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                          <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>
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<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>
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        <source src="https://static.igem.org/mediawiki/2018/f/fc/T--Vilnius-Lithuania--Fig7_Liposomes_formation_video.mp4" type="video/mp4">
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<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>
 
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<img src="https://static.igem.org/mediawiki/2018/4/4e/T--Vilnius-Lithuania--Fig8_Liposomes.png">
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<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>
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<img src="https://static.igem.org/mediawiki/2018/c/c2/T--Vilnius-Lithuania--Fig9_Liposomes.png">
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<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>
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<img src="https://static.igem.org/mediawiki/2018/3/34/T--Vilnius-Lithuania--Fig10_Liposomes.png"><p>
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<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:
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the group with a-hemolysin shows statistically significant (p< 0.0001) increase in fluorescence.</p>
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<p><a href="https://2018.igem.org/Team:Vilnius-Lithuania/Design#Liposomes">More about liposome production</a></p>
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<ul>
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    <li>Moreover, we proved that liposomes can incorporate one of the main membrane protein assembly machinery proteins BamA into their membranes.
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        </li>
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    </ul>
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    <p>When incubated in a solution consisting of soluble BAM complex components and liposomes, unfolded BamA (denatured in urea) was successfully reconstituted and therefore integrated into the liposome membranes. It demonstrates the activity of the BAM complex, because BamA cannot self-integrate into the lipid bilayer without the assistance of other proteins from Bam complex<sub>2</sub> (Fig. 6).
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<img src="https://static.igem.org/mediawiki/2018/6/6e/T--Vilnius-Lithuania--Simo_fig6.jpg">
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<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.
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    </p>
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<p><a href="https://2018.igem.org/Team:Vilnius-Lithuania/Design#BAM%20complex">More about Bam complex proteins</a>
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    </p>
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    <ul>
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            <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>
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        </ul>
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<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.
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<div class="image-container">
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<img src="https://static.igem.org/mediawiki/2018/4/47/T--Vilnius-Lithuania--Simui_bac_expo.jpg">
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<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>
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<p><a href="https://2018.igem.org/Team:Vilnius-Lithuania/Design#Surface_display_system">More about surface display</a>
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    <h2>References</h2>
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<ol>
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    <li>
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            Deshpande, S., Caspi, Y., Meijering, A. E. C. & Dekker, C. Octanol-assisted liposome assembly on chip. Nat. Commun. 7, 10447 (2016).
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    </li>
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    <li>Hagan, C. L., Westwood, D. B. & Kahne, D. Bam Lipoproteins Assemble BamA in Vitro. Biochemistry 52, 6108–6113 (2013).
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        </li>
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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.

Proof

The proof of SynDrop’s concept consists of several critical steps:

  • We have specified the parameters for cell-sized liposome (5-30 µm) synthesis by using our COMSOL model.

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.

Fig. 1 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.

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.

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) method11 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.

Fig. 2 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.

Fig. 3 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;

Fig. 4 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.

Fig. 5 a 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); b 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.

More about liposome production

  • Moreover, we proved that liposomes can incorporate one of the main membrane protein assembly machinery proteins BamA into their membranes.

When incubated in a solution consisting of soluble BAM complex components and liposomes, unfolded BamA (denatured in urea) was successfully reconstituted and therefore integrated into the liposome membranes. It demonstrates the activity of the BAM complex, because BamA cannot self-integrate into the lipid bilayer without the assistance of other proteins from Bam complex2 (Fig. 6).

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.

More about Bam complex proteins

  • 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.

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.

Fig. 7 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.

More about surface display

References

  1. Deshpande, S., Caspi, Y., Meijering, A. E. C. & Dekker, C. Octanol-assisted liposome assembly on chip. Nat. Commun. 7, 10447 (2016).
  2. Hagan, C. L., Westwood, D. B. & Kahne, D. Bam Lipoproteins Assemble BamA in Vitro. Biochemistry 52, 6108–6113 (2013).
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