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

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<H1>Proof of Concept</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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<h1 class="text-wall-heading">Proof of Concept</h1>
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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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<div class="text-wall-area-box">
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</ul>
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    <h2 class="text-wall-area-box-heading">The Composite Proof</h2>
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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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    <div class="scroll-area">
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    </p>
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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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</p>
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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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              </div>
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<ul>
 
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        <li>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.
 
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            </li>
 
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    </ul>
 
 
         </p>
 
         </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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        <button class="read-more-button">Read More</button>
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        <a class="pagination-anchor">
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            <div class="pagination-item"></div>
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            <span class="pagination-text">Description</span>
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    </div>
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</div>
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    <div class="modal-close"></div>
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    <div class="modal-content">
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        <h1>Description</h1>
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        <p></p>
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        <p></p>
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        <h2>What is SynORI?</h2>
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        <p>SynORI stands for synthetic origin of replication. It is a framework designed to make working with single
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            and multi-plasmid systems precise, easy and on top of that - more functional.</p>
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        <p>The SynORI framework enables scientists to build a multi-plasmid system in a standardized manner by:</p>
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        <ol>
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            <li>Selecting the number of plasmid groups</li>
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            <li>Choosing the copy number of each group</li>
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            <li>Picking the type of copy number control (specific to one group or regulating all of them at once).</li>
  
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<video width="100%" height="240" controls>
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         </ol>
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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>
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         </video>
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<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.
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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></p>
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<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;
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        <p>The framework also includes a possibility of adding a selection system that reduces the usage of antibiotics
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            (only 1 antibiotic for up to 5 different plasmids!) and an active partitioning system to make sure that low
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            copy number plasmid groups are not lost during the division.
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        </p>
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        <p></p>
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        <div class="img-cont">
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            <img src="https://static.igem.org/mediawiki/parts/8/84/Collect.png" alt="img">
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        <h2>Applications</h2>
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        <p>
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            <h5>Everyday lab work</h5>
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            <p>
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                A multi-plasmid system that is easy to assemble and control. With our framework the need to limit your
 +
                research to a particular plasmid copy number just because there are not enough right replicons to
 +
                choose from, is eliminated. With SynORI you can easily create a vector with a desired copy number that
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                suits your needs.</li>
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            </p>
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            <h5>Biological computing</h5>
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            <p>
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                The ability to choose a wide range of copy number options and their control types will make the
 +
                synthetic biology engineering much more flexible and predictable. Introduction of plasmid copy number
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                regulation is equivalent to adding a global parameter to a computer system. It enables the coordination
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                of multiple gene group expression.
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            </p>
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            <h5>Smart assembly of large protein complexes</h5>
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            <p>
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                The co-expression of multi-subunit complexes using different replicons brings incoherency to an already
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                chaotic cell system. This can be avoided by using SynORI, as in this framework every plasmid group uses
 +
                the same type of control, and in addition can act in a group-specific manner.</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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            <h5>Metabolic engineering</h5>
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<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.
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            <p>
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                A big challenge for heterologous expression of multiple gene pathways is to accurately adjust the
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                levels of each enzyme to achieve optimal production efficiency. Precise promoter tuning in
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                transcriptional control and synthetic ribosome binding sites in translational control are already
 +
                widely used to maintain expression levels. In addition to current approaches, our framework allows a
 +
                simultaneous multiple gene control. Furthermore, an inducible regulation that we offer, can make the
 +
                search for perfect conditions a lot easier.
  
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<img src="https://static.igem.org/mediawiki/2018/3/34/T--Vilnius-Lithuania--Fig10_Liposomes.png">
 
  
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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.
 
  
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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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            </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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        </p>
 
  
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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>
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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>
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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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        <table style="width:100%">
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    </p>
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<thead>
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     <ul>
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<td align='center'>Species sign in ODE system</td>
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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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<td align='center'>Species</td>
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        </ul>
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<td align='center'>Initial concentration (M)</td>
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</thead>
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<tbody>
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<tr>
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<td align='center'>A</td>
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<td align='center'>pDNA+RNA I+RNAII early</td>
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<td align='center'>0</td>
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</tr>
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<tr>
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<td align='center'>B</td>
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<td align='center'>pDNA+RNA II short</td>
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<td align='center'>0</td>
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</tr>
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<tr>
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<td align='center'>RNAI</td>
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<td align='center'>RNA I</td>
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<td align='center'>1E-6</td>
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</tr>
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<tr>
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<td align='center'>D</td>
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<td align='center'>pDNA+RNA II long</td>
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<td align='center'>0</td>
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</tr>
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<tr>
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<td align='center'>E</td>
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<td align='center'>pDNA+RNAII primer</td>
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<td align='center'>0</td>
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</tr>
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<tr>
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<td align='center'>F</td>
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<td align='center'>RNA II long</td>
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<td align='center'>0</td>
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</tr>
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<tr>
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<td align='center'>G</td>
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<td align='center'>pDNA</td>
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<td align='center'>4E-8*</td>
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</tr>
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<tr>
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<td align='center'>H</td>
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<td align='center'>pDNA+RNA II+RNA I late</td>
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<td align='center'>0</td>
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</tr>
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<tr>
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<td align='center'>RNA II</td>
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<td align='center'>RNA II</td>
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<td align='center'>0</td>
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</tr>
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<tr>
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<td align='center'>J</td>
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<td align='center'>RNAI+RNAII</td>
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<td align='center'>0</td>
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</tr>
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</tbody>
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</table>
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    </div>
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</div>
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                    </a>
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                </div>
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            <img src="https://static.igem.org/mediawiki/2018/5/5b/T--Vilnius-Lithuania--Invert-Icon.png"/>
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        <span class="invert-text">invert</span>
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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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<img src="https://static.igem.org/mediawiki/2018/4/47/T--Vilnius-Lithuania--Simui_bac_expo.jpg">
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<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.
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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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    </p>
 
  
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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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</ol>
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Revision as of 21:14, 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.

Description

What is SynORI?

SynORI stands for synthetic origin of replication. It is a framework designed to make working with single and multi-plasmid systems precise, easy and on top of that - more functional.

The SynORI framework enables scientists to build a multi-plasmid system in a standardized manner by:

  1. Selecting the number of plasmid groups
  2. Choosing the copy number of each group
  3. Picking the type of copy number control (specific to one group or regulating all of them at once).

The framework also includes a possibility of adding a selection system that reduces the usage of antibiotics (only 1 antibiotic for up to 5 different plasmids!) and an active partitioning system to make sure that low copy number plasmid groups are not lost during the division.

img

Applications

Everyday lab work

A multi-plasmid system that is easy to assemble and control. With our framework the need to limit your research to a particular plasmid copy number just because there are not enough right replicons to choose from, is eliminated. With SynORI you can easily create a vector with a desired copy number that suits your needs.

Biological computing

The ability to choose a wide range of copy number options and their control types will make the synthetic biology engineering much more flexible and predictable. Introduction of plasmid copy number regulation is equivalent to adding a global parameter to a computer system. It enables the coordination of multiple gene group expression.

Smart assembly of large protein complexes

The co-expression of multi-subunit complexes using different replicons brings incoherency to an already chaotic cell system. This can be avoided by using SynORI, as in this framework every plasmid group uses the same type of control, and in addition can act in a group-specific manner.

Metabolic engineering

A big challenge for heterologous expression of multiple gene pathways is to accurately adjust the levels of each enzyme to achieve optimal production efficiency. Precise promoter tuning in transcriptional control and synthetic ribosome binding sites in translational control are already widely used to maintain expression levels. In addition to current approaches, our framework allows a simultaneous multiple gene control. Furthermore, an inducible regulation that we offer, can make the search for perfect conditions a lot easier.

Species sign in ODE system Species Initial concentration (M)
A pDNA+RNA I+RNAII early 0
B pDNA+RNA II short 0
RNAI RNA I 1E-6
D pDNA+RNA II long 0
E pDNA+RNAII primer 0
F RNA II long 0
G pDNA 4E-8*
H pDNA+RNA II+RNA I late 0
RNA II RNA II 0
J RNAI+RNAII 0
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