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

 
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        <section class="design_subsections">
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      <section class="design_subsections">
            <h1 id="Liposomes">Liposomes</h1>
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      <h1 id="Liposomes">Liposomes</h1>
            <div class="third_level_links">
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      <div class="third_level_links">
              <a href="#Liposomes">Liposomes</a>
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          <a href="#Liposomes">Liposomes</a>
              <a href="#Ribosome_modifications">Ribosome modifications</a>
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          <a href="#Ribosome_modifications">Ribosome modifications</a>
              <a href="#BAM complex">BAM complex</a>
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          <a href="#BAM complex">BAM complex</a>
              <a href="#RNA_Thermoswitches">RNA Thermoswitches</a>
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          <a href="#RNA_Thermoswitches">RNA Thermoswitches</a>
              <a href="#Mistic_fusion_protein">Mistic fusion protein</a>
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          <a href="#Mistic_fusion_protein">Mistic fusion protein</a>
              <a href="#Surface_display_system">Surface display system</a>
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          <a href="#Surface_display_system">Surface display system</a>
             </div>
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        </div>
             <div>
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        <div>
              Lorem ipsum dolor, sit amet consectetur adipisicing elit. Esse beatae assumenda eaque ex recusandae pariatur sunt soluta modi facere laborum exercitationem odio iure magnam obcaecati quos voluptatibus placeat, ratione harum!
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             <h2>Liposomes as closed containers for bottom up research</h2>
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            <p></p>
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            <p>
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                    At the core of SynDrop lays a liposome. Liposomes are essentially synthetic vesicles, artificially synthesized droplets of liquid, separated from the environment by a lipid bilayer (Fig. 1). They act as containers that encapsulate purified transcriptional and translational machinery and other vital elements that enable complex circuitry design. They have become increasingly popular due to various applications such as being carriers for medicinal drugs<sup>1</sup>, closed environments for protein engineering<sup>2</sup> and characterization of RNAs<sup>3</sup>, as biosensors<sup>4</sup> and molecular diagnostic tools<sup>5</sup>. The growing perspectives of liposomes as scaffolds for synthetic circuitry and membrane protein research are compelling as they have a multitude of different parameters that can be controlled. These include size, composition of a lipid membrane and interior composition.
              Qui blanditiis, excepturi veritatis eaque temporibus voluptate maxime facere laborum voluptatem rerum ex a ipsum voluptatibus tempore, saepe sunt omnis nostrum sint? Voluptatum facilis omnis ea accusantium explicabo magnam architecto!
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            </p>
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            <p>
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                    <div class="image-container">
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                                      <img src="https://static.igem.org/mediawiki/2018/c/cc/T--Vilnius-Lithuania--Fig_1_NEW_su_uzrasu_Liposomes.png"/>
              Quae quam earum impedit laborum, nobis aspernatur fugit enim consequuntur provident laboriosam obcaecati doloremque ipsam quis modi quos ratione ipsum beatae? Voluptas, debitis eum! Dolorem accusantium et rem. Veniam, dicta!
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<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>
              Sequi aut, eos id nemo maiores iste! Dicta cum eos, incidunt aperiam voluptate facilis vero vel deleniti inventore accusantium cupiditate saepe dolore atque quisquam voluptates aliquam amet a! Hic, consectetur.
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                                  </p>
            </div>
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                    </div>
        </section>
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                    <h2>Requirements for liposomes</h2>
 +
             <p></p>
 +
            <p>
 +
                    There are many different ways to synthesize liposomes, resulting in a vast range of diverse outputs. For our project, we set particular requirements to suit our scientific design. Ideally, our liposomes must be stable, cell-sized, monodisperse, have a single bilayered membrane and demonstrate excellent encapsulation efficiency. The only approach to optimally combine these attributes proved to be microfluidics. In recent years, droplet-based microfluidic techniques have proved to achieve a robust control over physical parameters of the final product, therefore showing superiority compared to the macroscale bulk methods<sup>6</sup>. We successfully customized and integrated a novel octanol-assisted liposome assembly (OLA)<sup>7</sup> method for high-throughput production of our liposomes.
 +
            </p>
 +
            <p></p>
 +
            <h2>
 +
                    AutoCAD design for creating photomasks
 +
            </h2>
 +
            <p></p>
 +
            <p>
 +
                    The first step we had to take towards the production of liposomes was to design, fabricate and prepare a unique microfluidic device. A microfluidic channel design was created with the AutoCAD platform. The prototype acts as a photomask during the photolithography to create a master for the fabrication of microfluidic chips.  Our design consists of an array of 16 separate microfluidic channel devices distributed parallelly in groups of four on a single chip (Fig. 2). The dimensions of the microchannels limit the size range of the synthesized liposomes. To dig deeper into the details of how the dimensions of the microfluidic channels influence liposome size we created a phase-field based <strong><var><a href="https://2018.igem.org/Team:Vilnius-Lithuania/Model#COMSOL_model">SynFlow</a></var></strong> model with COMSOL Multiphysics. Auxiliary parametric sweeps were performed that defined the dimensions needed to attain cell – sized liposomes, a range from 5 µm to 30 µm. The final design can be downloaded here and used by anyone interested in the synthesis of liposomes.
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            </p>
 +
            <p>
 +
                    <div class="image-container">
 +
                                      <img src="https://static.igem.org/mediawiki/2018/0/0e/T--Vilnius-Lithuania--Fig2_Liposomes.png"/>
 +
                                      <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.</p>
 +
                                  </p>
 +
                    </div>
 +
                    <h2>Photolithography as a tool for microfluidic chip fabrication</h2>
 +
                    <p></p>
 +
                    <p>
 +
                            After calculating the exact parameters for microfluidic channels and receiving a printed photomask, photolithography is performed to create a master for microfluidic chip preparation. After completing this step, PDMS (<var>polydimethylsiloxane</var>) is poured on to the master left in a thermostat overnight. Inlets and outlets are punched with a biopsy puncher, and the PDMS is cleaned and plasma treated before attaching it to the PDMS coated microscope glass slides. Fig. 3 presents a simplified scheme demonstrating photolithography and other +6steps towards creating a microfluidic chip. To learn more details about the fabrication process, refer to <a href="https://2018.igem.org/Team:Vilnius-Lithuania/Protocols">our Protocols</a>. We called our chip LipoDrop.  The final form of LipoDrop is shown in Fig. 4. 
 +
                    </p>
 +
                            <div class="image-container">
 +
                                    <img src="https://static.igem.org/mediawiki/2018/7/7d/T--Vilnius-Lithuania--Fig3_Liposomes.png"/>
 +
                                    <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.</div></p>
 +
                        <div class="image-container">
 +
                                <img src="https://static.igem.org/mediawiki/2018/7/75/T--Vilnius-Lithuania--Fig4_Liposomes.png"/>
 +
                                <p><strong>Fig. 4 </strong> Final form of Lipodrop.</div>
 +
                    </p>
 +
                    <h2>Coating LipoDrop with PVA</h2>
 +
                    <p></p>
 +
                    <p>
 +
                            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.
 +
                    </p>
 +
                    <div class="image-container">
 +
                            <img src="https://static.igem.org/mediawiki/2018/2/29/T--Vilnius-Lithuania--Fig5_Liposomes.png"/>
 +
                            <p><strong>Fig. 5 </strong> A schematic representation of the interphase of air and PVA at the star shaped junction of LipoDrop.</p>
 +
                        </p>
 +
          </div>
 +
                    <h2>Lipovision software for fully automated microfluidic experiments</h2>
 +
                    <p></p>
 +
                    <p>
 +
                            In pursuit to substantially reduce the manual effort in performing coating procedure, we have designed a software called LipoVision. LipoVision software reduces human labor down to the bare minimum and optimizes the coating procedure entirely. The LipoVision software is based on the open standard computer library OpenCV and is written in Go. It is available on all operating systems and made to be accessible to any custom microfluidic experiment. Eventually, we are planning to apply this software for the fully automated synthesis of the liposomes. To learn more about LipoVision, refer to <a href="https://2018.igem.org/Team:Vilnius-Lithuania/Software">Software</a>.
 +
                    </p>
 +
                    <p></p>
 +
                    <h2>The synthesis of liposomes</h2>
 +
                    <p></p>
 +
                    <p>
 +
                            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.
 +
                    </p>
 +
                    <div class="image-container">
 +
                            <img src="https://static.igem.org/mediawiki/2018/7/77/T--Vilnius-Lithuania--Fig6_Liposomes.png"/>
 +
                            <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
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                            and integration of membrane proteins; <strong>LO</strong> phase consists of octanol and lipids that form a lipid bilayer; OA solution
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                            carries surfactants that stabilize the initial formation and propagation of the droplets along the microfluidic device.</p></div>
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                    <h2>Optimized flow rates for high throughput synthesis</h2>
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                    <p></p>
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                    <p>
 +
                            With this method, we effectively produce monodisperse bilayered liposomes with high throughput. It is important to note that together with the microchannel dimensions, the infusion rates of the different phases also have an impact on the size of the droplets. Additionally, the infusion rates regulate the throughput of liposome production. The already mentioned <strong><var>SynFlow</var></strong> model determines the optimized flow rates for the most stable and highest frequency synthesis. With the right dimensions and flow rates we reach the production rate that is up to 2000 Hz. The results suggest that this method could be successfully adapted for mass production of liposomes. Fig. 7 reveals the slowed down process of droplet formation.
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                    </p>
 +
                    <div class="image-container">
 +
<video width="50%" height="240" controls>
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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">
 +
</video>
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                            <p><strong>Fig. 7 </strong> High throughput formation of cell-sized liposomes. The video is 60x slowed down </p>   
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                    </p>
 +
                </div>
 +
 
 +
                            A simple liposome size frequency distribution was determined with an image analysis software ImageJ. A plugin SpotCaliper was utilized to identify circular objects and measure their diameters (Fig. 8a). Gaussian distribution was fitted to the frequency histogram. Results verify that the size of the liposomes follows the Gaussian distribution (Fig. 8b). It proves that the droplets are highly homogeneous. Average diameter of a liposome (results from a single batch experiment) is around 12 µm, with standard deviation of 0.4 µm which fits our requirements very well.
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                    </p>
 +
                    <div class="image-container">
 +
                            <img src="https://static.igem.org/mediawiki/2018/4/4e/T--Vilnius-Lithuania--Fig8_Liposomes.png"/>
 +
                            <p><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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                            diameter of each is measured; <strong>b</strong> size frequency distribution histogram fitted to Gaussian distribution (teal fit) proves
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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>                                   
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                    </p>
 +
                </div>                             
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                    </p>
 +
                    <h2>Characterization: encapsulation efficiency and internal synthesis</h2>
 +
                    <p></p>
 +
                    <p>
 +
                            To prove the encapsulation efficiency and test whether protein synthesis and folding are feasible within the liposomes we employed green fluorescent protein (GFP). First, plasmid DNA coding GFP (3.0 ng/µL) was encapsulated together with PURE<var>frex</var> 2.0 transcription translation system. The total reaction mixture was 40 µL. The microtubes were kept on ice during the production and collection of the liposomes. After all the reaction mixture was used up, the collected suspension was incubated in 37℃ for six hours. During the incubation time, negative and positive controls were prepared. The negative control contained the same reaction mixture as previously, excluding the DNA. Positive control, on the other hand, contained purified GFP proteins and no included DNA. The outer solution of all samples contained RNAse to ensure that no synthesis occurred outside the droplets (in case some liposomes erupted and released DNA to the outside). After the incubation period, fluorescent microscopy was utilized to reveal the results. Excitation and emission wavelengths of GFP are 488 nm and 510 nm, respectfully, therefore a suitable FITC filter was used.
 +
                    </p>
 +
                    <p>
 +
                            Brightfield images of the synthesized liposomes are presented in Fig. 9a. The same area was then imaged under the FITC filter Fig. 9b, showing prominent fluorescence. It confirms that the liposomes are biocompatible, and synthesis occurs within them successfully . These results, together with positive control (Fig. 9c) validated that the encapsulation efficiency is immensely effective, as all the liposomes exhibit fluorescence signal. Negative control exhibited no measurable fluorescence with FITC filter, as expected (data not shown), confirming that no contamination was present to distort the results.
 +
                    </p>
 +
                    <div class="image-container">
 +
                            <img src="https://static.igem.org/mediawiki/2018/c/c2/T--Vilnius-Lithuania--Fig9_Liposomes.png"/>
 +
                            <p><strong>Fig. 9 </strong> brightfield image of the liposomes that contain IVTT system and plasmid GFP DNA (after incubation);
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                            scale bar is 10 µm; <strong>b</strong> liposomes imaged with FITC: fluorescence confirms that transcription and translation
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                            reactions occur inside them; scale bar is 10 µm; <strong>c</strong> liposomes containing purified GFP protein: all the
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                            liposomes exhibit fluorescence validating excellent encapsulation efficiency; scale bar is 20 µm.</p>                                   
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                    </p>
 +
                </div> 
 +
                
 +
                    </p>
 +
                    <h2>Characterization: unilamellarity validation using α-hemolysin protein pores</h2>
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                    <p></p>
 +
                    <p>
 +
                            Most membrane proteins insert, fold and function properly only in unilamellar membranes, therefore unilamellarity is an absolutely critical parameter for studying MPs. To validate that our synthesized liposomes are truly unilamellar, we recruited the pore forming membrane protein α-hemolysin (2 mM) from Staphylococcus aureus. Monomers of α-hemolysin self-assemble to form mushroom-shaped heptameric 1.5 nm wide pores in the phospholipid bilayer. The integration of these proteins make the bilayer selectively permeable to small molecules sized 2 kDa or less<sup>8</sup>. This protein pore was chosen as it can perforate into only one bilayer, therefore a multilamellar membrane would not allow the internal content of the liposome to escape<sup>9</sup>. 
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                    </p>
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                    <p>
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                            To test the incorporation of α-hemolysin into the liposome membrane we used a membrane-impermeable fluorescent probe calcein. Calcein is a self quenching fluorophore - a unique molecule that exhibits fluorescence only after it is diluted. That means that if we encapsulate concentrated calcein solution inside of our liposomes, we should observe that while looking through a fluorescence filter they appear darker than the outer solution (Fig. 10a). That happens because a small fraction of liposomes inevitably falls apart, releasing calcein into the outside, thus diluting it and therefore we measure the fluorescence in the outer solution. Hypothetically, inserting α-hemolysin into the liposome membrane will increase the fluorescence of the outer solution to a much greater degree, as the calcein molecules can now leave the liposomes via the α-hemolysin pore.
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                    </p>
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                    <p>
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                            To test this hypothesis, we prepared two sets of liposomes. Lipid composition of liposomes was composed of DOPC and Cholesterol (cholesterol is necessary for the integration of α-hemolysin). Both sets of liposomes were prepared from the same batch microfluidic experiment. The same volume of liposomes was transferred into a solution with α-hemolysin and to an identical solution without the protein. Plate-reader measurements of fluorescence were then recorded, and results analyzed. As expected, in the absence of α-hemolysin (control), some background fluorescence was observed. However, in the solution where α-hemolysin was included, the measured fluorescence was significantly stronger (p < 0.0001 Fig. 10b) compared to the control group. That confirms the successful integration of α-hemolysin pore into the liposome membrane, explaining the increased fluorescence of the measured outer solution.
 +
                 
 +
                        </p>
 +
                        <div class="image-container">
 +
                                <img src="https://static.igem.org/mediawiki/2018/3/34/T--Vilnius-Lithuania--Fig10_Liposomes.png"/>
 +
                                <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
 +
                                a group with inserted α-hemolysin; nonparametrical Mann-Whitney U test was used for the statistical evaluation:
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                                the group with α-hemolysin shows statistically significant (p < 0.0001) increase in fluorescence</p>                                                 
 +
                        </p>
 +
                    </div> 
 +
                                           
 +
                    </p>
 +
                    <h3>References</h3>
 +
                    <p>
 +
                        <ol>
 +
                            <li>
 +
                                              Torchilin, V. P. Recent advances with liposomes as pharmaceutical carriers. Nat. Rev. Drug Discov. 4, 145–160 (2005).
 +
                            </li>
 +
                            <li>
 +
                                              Shin, J. & Noireaux, V. An E. coli cell-free expression toolbox: Application to synthetic gene circuits and artificial cells. ACS Synth. Biol. 1, 29–41 (2012).
 +
                            </li>
 +
                            <li>
 +
                                              Kapoor, M., Burgess, D. J. & Patil, S. D. Physicochemical characterization techniques for lipid based delivery systems for siRNA. Int. J. Pharm. 427, 35–57 (2012).
 +
                            </li>
 +
                            <li>
 +
                                              Michener, J. K., Thodey, K., Liang, J. C. & Smolke, C. D. Applications of genetically-encoded biosensors for the construction and control of biosynthetic pathways. Metab. Eng. 14, 212–222 (2012).
 +
                            </li>
 +
                            <li>
 +
                                              Pardee, K. et al. Paper-based synthetic gene networks. Cell 159, 940–954 (2014).
 +
                            </li>
 +
                            <li>
 +
                                              Carugo, D., Bottaro, E., Owen, J., Stride, E. & Nastruzzi, C. Liposome production by microfluidics: Potential and limiting factors. Sci. Rep. 6, (2016).
 +
                            </li>
 +
                            <li>
 +
                                              Deshpande, S., Caspi, Y., Meijering, A. E. C. & Dekker, C. Octanol-assisted liposome assembly on chip. Nat. Commun. 7, 1–9 (2016).
 +
                            </li>
 +
                            <li>
 +
                                              Noireaux, V. & Libchaber, A. A vesicle bioreactor as a step toward an artificial cell assembly. Proc. Natl. Acad. Sci. 101, 17669–17674 (2004).
 +
                            </li>
 +
                            <li>
 +
                                              Lu, L., Schertzer, J. W. & Chiarot, P. R. Continuous microfluidic fabrication of synthetic asymmetric vesicles. Lab Chip 15, 3591–3599 (2015).
 +
                            </li>
 +
                        </ol>
 +
                    </p>
 +
        </div>
 +
    </section>
 
         <section class="design_subsections">
 
         <section class="design_subsections">
 
         <h1 id="Ribosome_modifications">Ribosome modifications</h1>
 
         <h1 id="Ribosome_modifications">Ribosome modifications</h1>
Line 74: Line 230:
 
                     <div class="image-container">
 
                     <div class="image-container">
 
                     <img src="https://static.igem.org/mediawiki/2018/4/42/T--Vilnius-Lithuania--Fig1_Ribosomes.png"/>
 
                     <img src="https://static.igem.org/mediawiki/2018/4/42/T--Vilnius-Lithuania--Fig1_Ribosomes.png"/>
                    <strong>Fig. 1 </strong> Principle of ribosome attachment to the liposome membrane. The ribosome exit tunnel is localized near the membrane, resulting in transmembrane domains of newly synthesized peptides interacting with the membrane, reducing aggregation
+
                <p><strong>Fig. 1 </strong> Principle of ribosome attachment to the liposome membrane. The ribosome exit tunnel is localized near the membrane, resulting in transmembrane domains of newly synthesized peptides interacting with the membrane, reducing aggregation</p>
 
             </p>
 
             </p>
 
         </div>
 
         </div>
Line 97: Line 253:
 
                     <div class="image-container">
 
                     <div class="image-container">
 
                     <img src="https://static.igem.org/mediawiki/2018/f/fc/T--Vilnius-Lithuania--Fig2_Ribosomes.png"/>
 
                     <img src="https://static.igem.org/mediawiki/2018/f/fc/T--Vilnius-Lithuania--Fig2_Ribosomes.png"/>
                  <strong>Fig. 2 </strong> Scheme of the genome modification process:
+
                <p><strong>Fig. 2 </strong> Scheme of the genome modification process:
 
                   <ol>
 
                   <ol>
 
                       <li>
 
                       <li>
Line 111: Line 267:
 
                           4. pTargetF is cured and the process can be repeated with a new target.
 
                           4. pTargetF is cured and the process can be repeated with a new target.
 
                       </li>
 
                       </li>
                   </ol>
+
                   </ol></p>
            </p>
+
 
            <p>
+
 
                 </div>
 
                 </div>
                     For multi-gene editing, we chose to supply the donor sequence as a linear DNA strand (PCR product). Due to financial reasons, to construct the donor DNA sequence we performed separate PCRs of the homology arms (from the E. coli genome), selection marker (antibiotic resistance genes from available plasmids) (Fig. 4). The oligomers had the his and strep tag sequences incorporated into them alongside 2 different restriction sites. In case the distance between the ribosomes and the membrane wall was too small for our system to be efficient, we also designed alternative variants the would feature the his-tags connected via a highly flexible two-glycine-four-serine linker (GGSSSS), which is a highly popular linker for artificial fusion proteins.
+
                     <p>For multi-gene editing, we chose to supply the donor sequence as a linear DNA strand (PCR product). Due to financial reasons, to construct the donor DNA sequence we performed separate PCRs of the homology arms (from the E. coli genome), selection marker (antibiotic resistance genes from available plasmids) (Fig. 4). The oligomers had the his and strep tag sequences incorporated into them alongside 2 different restriction sites. In case the distance between the ribosomes and the membrane wall was too small for our system to be efficient, we also designed alternative variants the would feature the his-tags connected via a highly flexible two-glycine-four-serine linker (GGSSSS), which is a highly popular linker for artificial fusion proteins.
 
             </p>
 
             </p>
 
             <p>
 
             <p>
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                     <div class="image-container">
 
                     <div class="image-container">
 
                     <img src="https://static.igem.org/mediawiki/2018/a/a9/T--Vilnius-Lithuania--Fig3_Ribosomes.png"/>
 
                     <img src="https://static.igem.org/mediawiki/2018/a/a9/T--Vilnius-Lithuania--Fig3_Ribosomes.png"/>
                    <strong> Fig.3 </strong> Example of a constructed donor sequence. The sequence of the selected tag is present in primer used for the PCR of the homology arm that encompasses the target subunit. As a result, the tag sequence is fused to the ribosomal subunit gene.
+
                <p><strong> Fig.3 </strong> Example of a constructed donor sequence. The sequence of the selected tag is present in primer used for the PCR of the homology arm that encompasses the target subunit. As a result, the tag sequence is fused to the ribosomal subunit gene.</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/d/d5/T--Vilnius-Lithuania--Fig4_Ribosomes.png"/>
 
                     <img src="https://static.igem.org/mediawiki/2018/d/d5/T--Vilnius-Lithuania--Fig4_Ribosomes.png"/>
                    <strong> Fig. 4 </strong> PCR of homology arms, and antibiotic resistance genes
+
                <p><strong>Fig. 4 </strong> PCR of homology arms, and antibiotic resistance genes</p>
 
             </p>
 
             </p>
 
         </div>
 
         </div>
Line 136: Line 291:
 
                     <div class="image-container">
 
                     <div class="image-container">
 
                     <img src="https://static.igem.org/mediawiki/2018/8/86/T--Vilnius-Lithuania--Fig5_Ribosomes.png"/>
 
                     <img src="https://static.igem.org/mediawiki/2018/8/86/T--Vilnius-Lithuania--Fig5_Ribosomes.png"/>
                    <strong> Fig. 5 </strong> Constructed donor DNA sequences. The L29 donor DNA was not further revisited due to time constraints
+
                <p><strong>Fig. 5 </strong> Constructed donor DNA sequences. The L29 donor DNA was not further revisited due to time constraints</p>
 
             </p>
 
             </p>
 
         </div>
 
         </div>
 
             <p>
 
             <p>
                     The genome modifications were then carried according to <a href="https://2018.igem.org/Team:Vilnius-Lithuania/Protocols">Protocols</a> our protocol. Although cPCR gave us mixed results, we could not verify any colonies that afterwards grew on our selected marker antibiotics, and thus could not continue our experiments with them. It appears most likely that the genome modifications were not entirely successful, due to the somewhat unstable nature of the ligated linear DNA used for the donor sequence.
+
                     The genome modifications were then carried according to <a href="https://2018.igem.org/Team:Vilnius-Lithuania/Protocols">our protocol</a>. Although cPCR gave us mixed results, we could not verify any colonies that afterwards grew on our selected marker antibiotics, and thus could not continue our experiments with them. It appears most likely that the genome modifications were not entirely successful, due to the somewhat unstable nature of the ligated linear DNA used for the donor sequence.
 
             </p>
 
             </p>
 
             <p></p>
 
             <p></p>
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             </div>
 
             </div>
 
             <div>
 
             <div>
              Lorem ipsum dolor, sit amet consectetur adipisicing elit. Esse beatae assumenda eaque ex recusandae pariatur sunt soluta modi facere laborum exercitationem odio iure magnam obcaecati quos voluptatibus placeat, ratione harum!
+
            <h1>Background</h1>
              Provident, maxime ipsum veniam, rerum facere ad vero fugit ipsa natus recusandae sit voluptatum architecto laudantium vitae necessitatibus! Nesciunt illum porro sint odio sequi reprehenderit. Sint eligendi ex impedit recusandae!
+
<p>
              Alias obcaecati impedit iure recusandae quas asperiores tempore sint, consectetur veniam provident iste nulla fugit velit aliquam expedita, assumenda repellat dolorem dolore! Sit quis dolorem ad pariatur repellat reiciendis officiis.
+
</p>
              Asperiores molestiae eos quo inventore recusandae quae placeat delectus, natus sint. Ullam quas culpa nobis exercitationem omnis animi velit, deleniti fugit! Sapiente aperiam sit minima nostrum, rerum quae laudantium vero?
+
<P>Proteins that belong to a small group referred to as nonconstitutive membrane proteins can independently integrate into the membrane from the aqueous phase without the help of other proteins. This is because they possess a stable soluble form, that can bind to membranes and then insert and refold into another stable form.[1] However, most of the membrane proteins do not have a stable soluble form and rapidly aggregate when synthesized in the cytoplasm. That is why living organisms require additional machinery which facilitates MP insertion into membranes and catalyzes their folding.</P>
              Qui blanditiis, excepturi veritatis eaque temporibus voluptate maxime facere laborum voluptatem rerum ex a ipsum voluptatibus tempore, saepe sunt omnis nostrum sint? Voluptatum facilis omnis ea accusantium explicabo magnam architecto!
+
 
              Repellendus incidunt doloremque, a cum voluptates esse officia quia veniam architecto. Quibusdam deserunt nulla, dolore perspiciatis accusantium ad aliquam voluptatem iste iure, quae minus ipsa voluptates, sit voluptatum consequatur tempora?
+
<h2>Structure of BAM complex
              Architecto impedit in repellendus, quo dolorum sequi voluptatum omnis maxime perspiciatis aspernatur obcaecati sint iusto tenetur praesentium labore. Natus eveniet quaerat recusandae dignissimos nam sed distinctio quos fugiat aliquid eligendi.
+
</h2>
              Molestias inventore nobis minus aspernatur recusandae asperiores excepturi ipsam voluptas quos, quae voluptatem voluptatibus vero veniam dolores fuga aliquid neque ut dolorem beatae cum temporibus tempora? Iure expedita debitis corrupti.
+
<p>Assembly of the β-barrel bearing integral membrane proteins (MPs) into the target membrane is catalyzed by the β-barrel assembly machinery (BAM) complex. It contains five subunits, BamA–E.[2]  BamA is composed of a 16-strand β-barrel integral membrane part and a periplasmic domain, which consists of five globular subdomains called POTRA motifs that are essential for complex formation and interaction with a substrate β-barrel proteins.[3] BamB-E are lipoproteins, each attaching to the inner leaflet of the OM via an N-terminal lipid moiety and playing an important role in promoting the folding of OMP.[4] (Fig.1)
              Quae quam earum impedit laborum, nobis aspernatur fugit enim consequuntur provident laboriosam obcaecati doloremque ipsam quis modi quos ratione ipsum beatae? Voluptas, debitis eum! Dolorem accusantium et rem. Veniam, dicta!
+
</p>
              Sequi aut, eos id nemo maiores iste! Dicta cum eos, incidunt aperiam voluptate facilis vero vel deleniti inventore accusantium cupiditate saepe dolore atque quisquam voluptates aliquam amet a! Hic, consectetur.
+
<div class="image-container">
 +
    <img src="https://static.igem.org/mediawiki/2018/1/1c/T--Vilnius-Lithuania--Fig1_BAM_compl.png"/>
 +
<p><strong>Fig. 1</strong> Structure of BAM complex</p>
 +
</div>
 +
<h2>Outer membrane protein (OMP) insertion into membrane in bacteria cells
 +
</h2>
 +
<div class="image-container">
 +
    <img src="https://static.igem.org/mediawiki/2018/3/3a/T--Vilnius-Lithuania--Fig2_BAM_compl.png"/>
 +
<p><strong>Fig. 2</strong> 5 steps scheme of OMP insertion into OM.</p>
 +
</div>
 +
 
 +
<p>Generally MPs are integrated during translation with the assistance of the Sec Translocon. However, as there is no protein translation within the periplasm, OMPs require an alternative integration mechanism.  
 +
</p>
 +
<p>In the beginning, OMPs are synthesized in the cytoplasm with N-terminal signal sequence that directs them to the Sec translocon, which transfers OMPs through the inner membrane into periplasm.[5] (Fig.2, step 1)
 +
</p>
 +
<p>Integral membrane proteins forming β-barrel structures are prone to aggregate in aqueous environments. Therefore, after they transit a Sec channel, chaperones are required to bind OMPs to transfer them through the periplasmic compartment, while keeping them in an unfolded state to prevent aggregation.(Fig.2, step 2) The periplasmic chaperone, SurA has been shown to transfer most of the OMPs to the OM.[6] It has been shown that SurA directly participates in Bam-mediated OMP assembly by associating with BamA POTRA domain.[7] (Fig.2, step 3)</p>
 +
<p>Final folding takes place in BAM complex. However, the mechanism how BAM complex catalyzes the insertion of β-barrel proteins into the OM still remains not fully understood. Structure analysis implies that the cavity seems to be too small to accomodate a fully folded OMP substrate, even though it is large enough to house couple of substrate β-hairpins.[2] (Fig.2, step 4)Based on the structure, it has been suggested that the rotation of the ring-like structure by POTRA domains and lipoproteins leads to the opening of a junction between the first and last β-strands of the BamA β-barrel (lateral gate) which promotes the insertion of OMPs into the lipid bilayer.[8] (Fig. 2, step 5)
 +
</p>
 +
<h2>Our approach</h2>
 +
<p>In order to reconstitute the fully working Bam complex we relied on the mechanisms elucidated before. SurA is a periplasmic chaperone which binds to unfolded β-barrel proteins and retains them in unfolded state, thus preventing aggregation. BamB or BamD lipoproteins can bind BamA-SurA, direct the complex to the membrane, and catalyze BamA insertion as well as correct folding in the membrane. [9] (Fig. 3, step 1) Knowing that in bacteria most of the Bam lipoproteins are found in BAM complex and full five proteins complex can be purified with one tag without any cross-links, we expected high complex association constants among the components (Fig.3, step 2) and hypothesized that BAM complex could be assembled in vitro simply by encapsulating BamA-SurA associatives and Bam lipoproteins.</p>
 +
<div class="image-container">
 +
    <img src="https://static.igem.org/mediawiki/2018/3/30/T--Vilnius-Lithuania--Fig3_BAM_compl.png"/>
 +
<p><strong>Fig. 3</strong> Bam lipoproteins assemble BamA in vitro</p>
 +
</div>
 +
<p></p>
 +
<h1>Results</h1>
 +
<h2>Plasmid construction</h2>
 +
<p>To purify the components of BAM complex we have constructed 6 plasmids:</p>
 +
<p>
 +
    <ol>
 +
        <li>pET28b-BamA</li>
 +
        <li>pET22b-BamA</li>
 +
        <li>pET22b-BamB</li>
 +
        <li>pCDFDuet-BamC-BamD</li>
 +
        <li>pET22b-BamE</li>
 +
        <Li>pET28b-SurA(more details in protocols)</Li>
 +
    </ol>
 +
    which encode products of BamA-HisN6, BamA, BamB-HisC6, BamC, BamD, BamE-HisC8, SurA-HisN6. (Fig. 4 and Fig. 5)
 +
</p>
 +
    <div class="image-container">
 +
            <img src="https://static.igem.org/mediawiki/2018/5/54/T--Vilnius-Lithuania--Fig4.1_BAM_compl.png"/>
 +
                <img src="https://static.igem.org/mediawiki/2018/3/3d/T--Vilnius-Lithuania--Fig4.2_BAM_compl.png"/>
 +
<p><strong>Fig. 4</strong> Maps of constructed plasmids</p>
 +
        <img src="https://static.igem.org/mediawiki/2018/2/2e/T--Vilnius-Lithuania--Fig5_BAM_compl.png"/>
 +
<p><strong>Fig. 5</strong> PCR products of genes of BAM complex 1,18 – GeneRuler 1 kb DNA ladder;  4,5 – SurA (1250bp); 6,7 -  BamE (390bp); 8,9 – BamB (1201bp); 10, 11 – BamC (1056bp); 12, 13 – BamD(756bp); 14,16 – BamA (2455bp)</p>
 +
</div>
 +
            <h2>
 +
    Protein purification
 +
</h2>
 +
<p>We relied on three different strategies to purify the separate BAM complex components: </p>
 +
<ul>
 +
    <li>For our experiments we needed an unfolded BamA. Therefore, we overexpressed BamA , which we isolated in the form of inclusion bodies and then dissolved in 8M urea without any further purification steps.</li>
 +
</ul>
 +
<div class="image-container">
 +
    <img src="https://static.igem.org/mediawiki/2018/8/85/T--Vilnius-Lithuania--Fig6_BAM_compl.png"/>
 +
<p><strong>Fig. 6</strong> BamA after purification
 +
    1 – BamA, 2 – BamA-HisN6, L – PageRuler Unstained Broad Range Protein Ladder</p>
 +
</div>
 +
    <ul>
 +
<Li>Bam B-D lipoproteins were expressed with the pelB signal sequence, leading them to be exported to the periplasm where lipidation takes place. We then isolated the proteins from the membrane fraction, which we solubilised with specific detergents before purification using Ni-NTA (Fig.7 and Fig.9) and gelfiltration (size-exclusion) (Fig. 8 and Fig. 9) columns. BamCDE were purified as a single subcomplex via one octahistidine tag on BamE.</Li>
 +
</ul>
 +
<h3>After purification with Ni-NTA column:</h3>
 +
<p>
 +
<div class="image-container">
 +
    <img src="https://static.igem.org/mediawiki/2018/2/2c/T--Vilnius-Lithuania--Fig7_BAM_compl.png"/>
 +
</p>
 +
<p><strong>Fig. 7</strong> BamB after Ni-NTA column purification Lane L – PageRuler Unstained Protein Ladder, Lane 1 – Sample  loaded on Ni-NTA Column,
 +
    Lanes 2-3 – Flow through fractions, Lanes 4-5 – washing fractions, Lanes 6-9 – Elution fractions
 +
    </p>
 +
</div>
 +
<h3> After gelfiltration:</h3>
 +
 
 +
<div class="image-container">
 +
<p>
 +
    <img src="https://static.igem.org/mediawiki/2018/2/2e/T--Vilnius-Lithuania--Fig8_BAM_compl.png"/>
 +
</p>
 +
<p><strong>Fig. 8</strong> BamB fractions after gelfiltration
 +
Lane L – PageRuler Unstained Protein Ladder, Lanes 1-14 Elution fractions
 +
</p>
 +
</div>
 +
<h3> After gelfiltration:</h3>
 +
 
 +
<div class="image-container">
 +
 
 +
<p>
 +
    <img src="https://static.igem.org/mediawiki/2018/3/36/T--Vilnius-Lithuania--Fig9_BAM_compl.png"/>
 +
</p>
 +
<p><strong>Fig. 9</strong> BamCDE after Ni-NTA column purification Lane L – PageRuler Unstained Protein Ladder, Lane 1,2 - Flow through fractions, Lanes 3-4 – washing fractions, Lanes 5-7 – Elution fractions
 +
</p>
 +
<div>
 +
<h3>After gelfiltration:</h3>
 +
 
 +
<div class="image-container">
 +
 
 +
<p>
 +
    <img src="https://static.igem.org/mediawiki/2018/a/ae/T--Vilnius-Lithuania--Fig10_BAM_compl.png"/>
 +
</p>
 +
<p><strong>Fig. 10</strong> BamCDE fractions after gelfiltration
 +
    Lane 1 – PageRuler Unstained Protein Ladder,  2-15 elution fractions</p>
 +
</div>
 +
 
 +
<ul>
 +
    <li>
 +
        While SurA is a periplasmic protein, we had no issues overexpressing it in the cytoplasm for increased yield. We used a hexahistidine tag and purified using Ni-NTA (Fig.11) and gelfiltration (Fig.12) columns.
 +
    </li>
 +
</ul>
 +
<h3>After Ni-NTA column:</h3>
 +
<div class="image-container">
 +
<img src="https://static.igem.org/mediawiki/2018/3/33/T--Vilnius-Lithuania--Fig11_BAM_compl.png"/>
 +
<p><strong>Fig. 11 </strong>SurA after purification with Ni-NTA column
 +
    L - PageRuler Unstained Protein Ladder, 1 - Protein loaded on Ni-NTA Column, Lane 2 – Flow through fraction, 3 - washing fraction, 4-9 elution fractions
 +
    </p>
 +
</div>
 +
<h3>After gelfiltration</h3>
 +
<div class="image-container">
 +
<p>
 +
    <img src="https://static.igem.org/mediawiki/2018/e/e3/T--Vilnius-Lithuania--Fig12_BAM_compl.png"/>
 +
</p>
 +
<p><strong>Fig. 12 </strong>SurA fractions after gelfiltration
 +
    L - PageRuler Unstained Protein Ladder, 1-9 elution fractions
 +
    </p>
 +
</div>
 +
<H2>Folding assay</H2>
 +
<p>To determine whether the purified proteins act as expected we conducted a specific a folding assay. Proteins possessing β-barrel structures exhibit a unique characteristic - when mixed with SDS (for SDS-PAGE) but unboiled, the β-barrel structure remains intact, which causes the protein to move differently in the SDS-PAGE gel in comparison to the same protein lacking these structures - which occurs when it is denatured or did not originally fold. Exploiting this characteristic makes it possible to observe and quantify protein folding levels.</p>
 +
<p>For the first experiment we observed if BamB and BamCDE can incorporate the unfolded BamA protein into the membrane and reconstitute the complete BAM complex. This was accomplished by incubating SurA with BamA denatured in urea, then transferring it into a solution featuring liposomes, BamB and BamCDE,  then further incubating for 2 hours. As BamA was expressed with a his-tag, we performed a blot to determine the level of protein folding (Fig. 13). </p>
 +
<div class="image-container">
 +
<p>
 +
    <img src="https://static.igem.org/mediawiki/2018/7/73/T--Vilnius-Lithuania--Fig13_BAM_compl.png"/>
 +
</p>
 +
<p><strong>Fig. 13</strong> Western blot of BamA folding. 1U - sample 1 unboiled, 1B - sample 1 boiled, 2U - sample 2 unboiled, 2B - sample 2 boiled, L - ladder
 +
</p>
 +
</div>
 +
<p>As we can see from the results, after only 2 hours of incubation over 50% of BamA was processed and correctly folded, which is an indicator of proper functionality. At higher concentrations or within more enclosed environments, such as encapsulated within the liposome, efficiency is bound to increase.</p>
 +
 
 +
<h1>Conclusions</h1>
 +
<p></p>
 +
<p>We managed to successfully isolate the BAM protein complex at a high purity with relatively few steps. The BAM complex not only shows activity in vitro, it shows efficient activity within the presence of liposomes, which shows that it is functional and suitable for our system.
 +
</p>
 +
<p></p>
 +
<h1>Discussion</h1>
 +
 
 +
 
 +
<p></p>
 +
<p>The study of MPs is and continues to be a difficult area of study, due to the sheer difficulty in handling them. As we have emphasized before, the integration of MP’s is a particularly pronounced issue, being borderline impossible for some cases in vitro. However, we have managed to demonstrate, that with our newly designed approach utilizing the BAM complex, the SynDrop system can allow for much more efficient insertion of MP’s as well as greatly expanding the total amount of viable MP’s for high throughput in vitro studies.</p>
 +
<h2>References</h2>
 +
<p>
 +
    <ol>
 +
        <Li>White, S. & Wimley, W. MEMBRANE PROTEIN FOLDING AND STABILITY: Physical Principles. Annual Review of Biophysics and Biomolecular Structure 28, 319-365 (1999).</Li>
 +
    <li>Noinaj, N., Rollauer, S. & Buchanan, S. The β-barrel membrane protein insertase machinery from Gram-negative bacteria. Current Opinion in Structural Biology 31, 35-42 (2015).
 +
    </li>
 +
    <li>Fleming, P. et al. BamA POTRA Domain Interacts with a Native Lipid Membrane Surface. Biophysical Journal 110, 2698-2709 (2016).
 +
    </li>
 +
    <li>Hussain, S. & Bernstein, H. The Bam complex catalyzes efficient insertion of bacterial outer membrane proteins into membrane vesicles of variable lipid composition. Journal of Biological Chemistry 293, 2959-2973 (2018).
 +
    </li>
 +
    <li>Driessen, A. & Nouwen, N. Protein Translocation Across the Bacterial Cytoplasmic Membrane. Annual Review of Biochemistry 77, 643-667 (2008).
 +
    </li>
 +
    <li>Sklar, J., Wu, T., Kahne, D. & Silhavy, T. Defining the roles of the periplasmic chaperones SurA, Skp, and DegP in Escherichia coli. Genes &amp; Development 21, 2473-2484 (2007).
 +
    </li>
 +
    <li> Bennion, D., Charlson, E., Coon, E. & Misra, R. Dissection of β-barrel outer membrane protein assembly pathways through characterizing BamA POTRA 1 mutants of Escherichia coli. Molecular Microbiology 77, 1153-1171 (2010).
 +
    </li>
 +
    <li>Gu, Y. et al. Structural basis of outer membrane protein insertion by the BAM complex. Nature 531, 64-69 (2016).
 +
    </li>
 +
    <li>Hagan, C., Westwood, D. & Kahne, D. Bam Lipoproteins Assemble BamA in Vitro. Biochemistry 52, 6108-6113 (2013).
 +
    </li>
 +
   
 +
    </ol>
 +
</p>-
 
             </div>
 
             </div>
 
         </section>
 
         </section>
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               <p>
 
               <p>
 
                   <img src="https://static.igem.org/mediawiki/2018/c/ca/T--Vilnius-Lithuania--THERMO_fig_2.png"/>
 
                   <img src="https://static.igem.org/mediawiki/2018/c/ca/T--Vilnius-Lithuania--THERMO_fig_2.png"/>
                  <strong>Fig. 2</strong> Electrophoresis gel of PCR products: 6 - Sw2, 7 - Sw3, 8 - Sw6, 9 - Sw7, 10 - Sw9, 11 - Sw11.
+
                <p><strong>Fig. 2 </strong> Electrophoresis gel of PCR products: 6 - Sw2, 7 - Sw3, 8 - Sw6, 9 - Sw7, 10 - Sw9, 11 - Sw11.</p>
 
               </p>
 
               </p>
 
               <p>
 
               <p>
                   pRSET plasmid and Sw<sub>x</sub> PCR products were digested with restriction enzymes and ligated, while GJ<sub>x</sub> PCR products were phosphorylated and ligated to produce plasmids from linear products. DH5α competent cells were transformed and plated on lysogeny broth (LB) media with ampicillin (Amp) and grown for 16 hours. Positive colonies were selected by colony PCR or restriction analysis (Fig. 3 and Fig. 4) and grown in 5 mL LB media. Plasmids were purified and BL21 competent cells were transformed. Three tubes of every construct plus plasmid with GFP without RNA thermometer were grown till OD<sub>600</sub> reached 0.4. Control samples were taken and protein expression was induced with Isopropyl β-D-1-thiogalactopyranoside (IPTG). One tube of every construct was grown in 24 ˚C, 30 ˚C, and 37 ˚C. Samples were taken after 1 and 2 hours. SDS-PAGE was run (for elaborate protocol see Notebook/<a href="https://2018.igem.org/Team:Vilnius-Lithuania/Protocols">Protocols</a>). Fig. 5, Fig 6 and Fig. 7 depicts GFP expression at different temperatures. Although our RNA thermometers were designed to melt at 37 ˚C, some displayed leakiness to different extent. GJ3 (link:<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2622011">BBa_K2622011</a>) RNA thermometer was the leakeast and allowed for GFP translation at lower temperatures. On the other hand, when grown at 37 ˚C, it unlocked the translation of GFP to highest yields. GJ2 (link:<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2622010">BBa_K2622010</a>) was less leaky, but inhibited protein translation more strictly when grown at 37 ˚C. GJ6 (link: <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2622012">BBa_K2622012</a>), GJ9 (link:<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2622013">BBa_K2622013</a>), and GJ10 (link: <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2622014">BBa_K2622014</a>) suppressed GFP production at 24 ˚C and 30 ˚C at similar level. They also inhibited translation to some extent at higher temperatures, meaning their melting temperature was not reached. Altogether these results prove, that our synthetic thermoswitches are temperature-responsive and act in physiological temperature range needed for IVTT reaction and also for BamA folding and membrane insertion.
+
                   pRSET plasmid and Sw<sub>x</sub> PCR products were digested with restriction enzymes and ligated, while GJ<sub>x</sub> PCR products were phosphorylated and ligated to produce plasmids from linear products. DH5α competent cells were transformed and plated on lysogeny broth (LB) media with ampicillin (Amp) and grown for 16 hours. Positive colonies were selected by colony PCR or restriction analysis (Fig. 3 and Fig. 4) and grown in 5 mL LB media. Plasmids were purified and BL21 competent cells were transformed. Three tubes of every construct plus plasmid with GFP without RNA thermometer were grown till OD<sub>600</sub> reached 0.4. Control samples were taken and protein expression was induced with Isopropyl β-D-1-thiogalactopyranoside (IPTG). One tube of every construct was grown in 24 ˚C, 30 ˚C, and 37 ˚C. Samples were taken after 1 and 2 hours. SDS-PAGE was run (for elaborate protocol see <a href="https://2018.igem.org/Team:Vilnius-Lithuania/LabBook">Notebook</a>/<a href="https://2018.igem.org/Team:Vilnius-Lithuania/Protocols">Protocols</a>). Fig. 5, Fig. 6 and Fig. 7 depicts GFP expression at different temperatures. Although our RNA thermometers were designed to melt at 37 ˚C, some displayed leakiness to different extent. GJ3 (link:<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2622011">BBa_K2622011</a>) RNA thermometer was the leakeast and allowed for GFP translation at lower temperatures. On the other hand, when grown at 37 ˚C, it unlocked the translation of GFP to highest yields. GJ2 (link:<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2622010">BBa_K2622010</a>) was less leaky, but inhibited protein translation more strictly when grown at 37 ˚C. GJ6 (link: <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2622012">BBa_K2622012</a>), GJ9 (link:<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2622013">BBa_K2622013</a>), and GJ10 (link: <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2622014">BBa_K2622014</a>) suppressed GFP production at 24 ˚C and 30 ˚C at similar level. They also inhibited translation to some extent at higher temperatures, meaning their melting temperature was not reached. Altogether these results prove, that our synthetic thermoswitches are temperature-responsive and act in physiological temperature range needed for IVTT reaction and also for BamA folding and membrane insertion.
 
               </p>
 
               </p>
 
               <p>
 
               <p>
 
                   <img src="https://static.igem.org/mediawiki/2018/d/d8/T--Vilnius-Lithuania--THERMO_fig_3.png"/>
 
                   <img src="https://static.igem.org/mediawiki/2018/d/d8/T--Vilnius-Lithuania--THERMO_fig_3.png"/>
                  <strong>Fig. 3</strong> Restriction analysis of GJ<sub>x</sub> constructs
+
                <p><strong>Fig. 3 </strong> Restriction analysis of GJ<sub>x</sub> constructs</p>
 
               </p>
 
               </p>
 
               <p>
 
               <p>
 
                   <img src="https://static.igem.org/mediawiki/2018/a/a4/T--Vilnius-Lithuania--THERMO_fig_4.png"/>
 
                   <img src="https://static.igem.org/mediawiki/2018/a/a4/T--Vilnius-Lithuania--THERMO_fig_4.png"/>
                  <strong>Fig. 4</strong> Colony PCR of RNA thermometers in pSB1C3 plasmid.
+
                <p><strong>Fig. 4 </strong> Colony PCR of RNA thermometers in pSB1C3 plasmid.</p>
 
               </p>
 
               </p>
 
               <p>
 
               <p>
 
                   <img src="https://static.igem.org/mediawiki/2018/4/46/T--Vilnius-Lithuania--THERMO_fig_5.png"/>
 
                   <img src="https://static.igem.org/mediawiki/2018/4/46/T--Vilnius-Lithuania--THERMO_fig_5.png"/>
                  <strong>Fig. 5</strong> expression at 24 ˚C. On the right you can see GFP expression without RNA thermometer.  
+
                <p><strong>Fig. 5 </strong> expression at 24 ˚C. On the right you can see GFP expression without RNA thermometer.</p>
 
               </p>
 
               </p>
               <p>
+
               <p> <div class="image-container">
 
                   <img src="https://static.igem.org/mediawiki/2018/d/dd/T--Vilnius-Lithuania--THERMO_fig_6.png"/>
 
                   <img src="https://static.igem.org/mediawiki/2018/d/dd/T--Vilnius-Lithuania--THERMO_fig_6.png"/>
                  <strong>Fig. 6</strong> GFP expression at 30 ˚C. On the right you can see GFP expression without RNA thermometer.
+
                <p><strong>Fig. 6 </strong> GFP expression at 30 ˚C. On the right you can see GFP expression without RNA thermometer.</p>
 
               </p>
 
               </p>
               <p>
+
</div>
 +
               <p>  
 +
<div class="image-container">
 +
 
 
                   <img src="https://static.igem.org/mediawiki/2018/7/78/T--Vilnius-Lithuania--THERMO_fig_7.png"/>
 
                   <img src="https://static.igem.org/mediawiki/2018/7/78/T--Vilnius-Lithuania--THERMO_fig_7.png"/>
                  <strong>Fig. 7</strong> GFP expression in 37 ˚C. On the right you can see GFP expression without RNA thermometer.
+
                <p><strong>Fig. 7 </strong> GFP expression in 37 ˚C. On the right you can see GFP expression without RNA thermometer.</p>
 
               </p>
 
               </p>
 +
</div>
 
               <p></p>
 
               <p></p>
 
               <h1>Discussion</h1>
 
               <h1>Discussion</h1>
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               </p>
 
               </p>
 
               <p>
 
               <p>
 +
<div class="image-container">
 
                   <img src="https://static.igem.org/mediawiki/2018/a/af/T--Vilnius-Lithuania--Fig8_NEW_thermoswitches.png"/>
 
                   <img src="https://static.igem.org/mediawiki/2018/a/af/T--Vilnius-Lithuania--Fig8_NEW_thermoswitches.png"/>
                  <strong>Fig. 8</strong> Associational scheme of thermoswitches’ action in the SynDrop system. Not locking the concomitant translation of our target protein and BamA results in target protein aggregation due to insufficient membrane insertion and  assembling potential of BamA.
+
                <p><strong>Fig. 8</strong> Associational scheme of thermoswitches’ action in the SynDrop system. Not locking the concomitant translation of our target protein and BamA results in target protein aggregation due to insufficient membrane insertion and  assembling potential of BamA.</p>
 
               </p>
 
               </p>
 +
</div>
 
               <p>
 
               <p>
 +
<div class="image-container">
 
                   <img src="https://static.igem.org/mediawiki/2018/8/8b/T--Vilnius-Lithuania--Fig9_NEW_thermoswitches.png"/>
 
                   <img src="https://static.igem.org/mediawiki/2018/8/8b/T--Vilnius-Lithuania--Fig9_NEW_thermoswitches.png"/>
                  <strong>Fig. 9</strong> Associational scheme of thermoswitches’ action in the SynDrop system. Locking up translation gives time for proper folding and insertion of BamA and prevents undesirable aggregation of target membrane proteins.
+
                <p><strong>Fig. 9</strong> Associational scheme of thermoswitches’ action in the SynDrop system. Locking up translation gives time for proper folding and insertion of BamA and prevents undesirable aggregation of target membrane proteins.</p>
 
               </p>
 
               </p>
 +
</div>
 
               <p>
 
               <p>
 
                   Additionally, while creating SynDrop, we have considered various options on how to make our complex cell-free system more user-controllable and predictable. Cell-free systems are becoming an attractive platform for <var>in vitro</var> compartmentalization and protein research, and although usually compositionally sensitive, they also offer a platform for building synthetic genetic regulatory tools or logic gates. Both the need to control the translation time of target genes and desire to provide more modularity for our synthetic system, led us to exploring RNA thermometers as a viable option to perform these tasks. They have minimal molecular burden and are easy to modulate. These properties encouraged us to developed a library of synthetic RNA thermometers suitable to translationally regulate the expression of our fusion constructs in bacteria with a further possibility to transfer them to IVTT systems. All of the RNA thermometers including those we found in literature and our <var>de novo</var> modelled ones were optimized for best performance at 37<sup>o</sup>C, bearing in mind their future transition to IVTT system, whose optimum performance temperature is also 37<sup>o</sup>C. Consequently, our experiments showed that our synthetic RNA thermometers, despite their simplistic structures compared to naturally occurring ones, efficiently triggered the expression of target constructs at 37<sup>o</sup>C, and successfully locked it at lower temperatures having made them an ideal complement to our liposome IVTT system. All of our thermoswitches unlocked the expression to similarly high levels at 37<sup>o</sup>C, but differed in terms of leakiness and success at inhibiting translation at lower temperatures.
 
                   Additionally, while creating SynDrop, we have considered various options on how to make our complex cell-free system more user-controllable and predictable. Cell-free systems are becoming an attractive platform for <var>in vitro</var> compartmentalization and protein research, and although usually compositionally sensitive, they also offer a platform for building synthetic genetic regulatory tools or logic gates. Both the need to control the translation time of target genes and desire to provide more modularity for our synthetic system, led us to exploring RNA thermometers as a viable option to perform these tasks. They have minimal molecular burden and are easy to modulate. These properties encouraged us to developed a library of synthetic RNA thermometers suitable to translationally regulate the expression of our fusion constructs in bacteria with a further possibility to transfer them to IVTT systems. All of the RNA thermometers including those we found in literature and our <var>de novo</var> modelled ones were optimized for best performance at 37<sup>o</sup>C, bearing in mind their future transition to IVTT system, whose optimum performance temperature is also 37<sup>o</sup>C. Consequently, our experiments showed that our synthetic RNA thermometers, despite their simplistic structures compared to naturally occurring ones, efficiently triggered the expression of target constructs at 37<sup>o</sup>C, and successfully locked it at lower temperatures having made them an ideal complement to our liposome IVTT system. All of our thermoswitches unlocked the expression to similarly high levels at 37<sup>o</sup>C, but differed in terms of leakiness and success at inhibiting translation at lower temperatures.
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               </p>
 
               </p>
 
               <p>
 
               <p>
                   MstX comprises 110 residues that are arranged into a four-helix bundle exposing numerous polar and charged amino acids (Fig. 1). This α-helical protein is characterized by an uncommonly hydrophilic surface. Until this day there is a great debate on how MstX is able to autonomously associate with a lipid bilayer despite its hydrophilic surface <sup>2</sup>. It is known that three of the four MstX helices are much shorter than transmembrane helices of canonical integral MPs. In general, the four helices of this protein show no apparent differences in hydrophobicity or charge distribution among each other.  
+
                   MstX comprises 110 residues that are arranged into a four-helix bundle exposing numerous polar and charged amino acids. This α-helical protein is characterized by an uncommonly hydrophilic surface. Until this day there is a great debate on how MstX is able to autonomously associate with a lipid bilayer despite its hydrophilic surface <sup>2</sup>. It is known that three of the four MstX helices are much shorter than transmembrane helices of canonical integral MPs. In general, the four helices of this protein show no apparent differences in hydrophobicity or charge distribution among each other.  
 
               </p>
 
               </p>
              <p>
+
         
                    <div class="image-container">
+
                            <img src="https://static.igem.org/mediawiki/2018/d/d1/T--Vilnius-Lithuania--Fig1_Mistic.png"/>
+
                            <p><strong>Fig. 1 </strong> NMR structure of Mistic (MstX). Protein is comprised of four ɑ-helices with a polar lipid-facing surface. Topology measurements have shown that both C-terminus and N-terminus of MstX are exposed at the same side. Adapted by Yarnell, 2005</p>
+
              </div>
+
 
               <p>
 
               <p>
 
                   MstX was identified back in 2005 by Rooslid and colleagues. Interestingly, until this day little is known about how MstX promotes integral protein targeting to the membrane<sup>3</sup>. Recently it has found a novel application as a fusion tag supporting the recombinant production and bilayer insertion of other membrane proteins (MPs)<sup>1</sup>. MstX, when fused to the N-terminus of integral MPs, enables the cargo proteins to fold into their native conformations in the membrane, thus yielding high-level expression. It is known that MstX autonomously targets proteins to the membrane bypassing the canonical secretory apparatus, like Sec translocon. In addition to this, it was indirectly presumed that MstX lacks any recognizable signal sequence <sup>2</sup>.  
 
                   MstX was identified back in 2005 by Rooslid and colleagues. Interestingly, until this day little is known about how MstX promotes integral protein targeting to the membrane<sup>3</sup>. Recently it has found a novel application as a fusion tag supporting the recombinant production and bilayer insertion of other membrane proteins (MPs)<sup>1</sup>. MstX, when fused to the N-terminus of integral MPs, enables the cargo proteins to fold into their native conformations in the membrane, thus yielding high-level expression. It is known that MstX autonomously targets proteins to the membrane bypassing the canonical secretory apparatus, like Sec translocon. In addition to this, it was indirectly presumed that MstX lacks any recognizable signal sequence <sup>2</sup>.  
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               </p>
 
               </p>
 
               <p>
 
               <p>
                   First of all, it was concluded that MstX-OmpA-X and OmpA-X (X - additional part of the construct) are expressed in cells (Fig. 2).  
+
                   First of all, it was concluded that MstX-OmpA-X and OmpA-X (X - additional part of the construct) are expressed in cells (Fig. 1).  
 
               </p>
 
               </p>
 
               <p>
 
               <p>
 
                     <div class="image-container">
 
                     <div class="image-container">
 
                             <img src="https://static.igem.org/mediawiki/2018/f/fa/T--Vilnius-Lithuania--Fig2_Mistic.png"/>
 
                             <img src="https://static.igem.org/mediawiki/2018/f/fa/T--Vilnius-Lithuania--Fig2_Mistic.png"/>
                             <p><strong>Fig. 2 </strong> OmpA-X and MstX-OmpA-X (X - additional part of the construct; shown with arrows) expression in <var>E. coli</var>; SDS-PAGE after induction with IPTG for 2 hours and 4 hours; K - control, M - protein ladder </p>
+
                             <p><strong>Fig. 1 </strong> OmpA-X and MstX-OmpA-X (X - additional part of the construct; shown with arrows) expression in <var>E. coli</var>; SDS-PAGE after induction with IPTG for 2 hours and 4 hours; K - control, M - protein ladder </p>
 
             </div>
 
             </div>
 
              
 
              
                   Also, we checked if we could fuse MstX with other integral membrane proteins. In this case, IgA protein (Fig. 3).  
+
                   <p>Also, we checked if we could fuse MstX with other integral membrane proteins. In this case, IgA protein (Fig. 3).  
 
               </p>
 
               </p>
 
                     <p>
 
                     <p>
 
                             <div class="image-container">
 
                             <div class="image-container">
 
                                     <img src="https://static.igem.org/mediawiki/2018/3/31/T--Vilnius-Lithuania--Fig3_Mistic.png"/>
 
                                     <img src="https://static.igem.org/mediawiki/2018/3/31/T--Vilnius-Lithuania--Fig3_Mistic.png"/>
                                     <p><strong>Fig. 3 </strong> IgA, IgA-MstX, and X-IgA-MstX (X - additional part of the construct; shown with arrows) expression in <var>E. coli</var>; SDS-PAGE; U4-2 - samples affected with 8M urea, S2-4 - samples affected with protein denaturation dye, K - control, M - protein ladder </p>
+
                                     <p><strong>Fig. 2 </strong> IgA, IgA-MstX, and X-IgA-MstX (X - additional part of the construct; shown with arrows) expression in <var>E. coli</var>; SDS-PAGE; U4-2 - samples affected with 8M urea, S2-4 - samples affected with protein denaturation dye, K - control, M - protein ladder </p>
 
                     </div>
 
                     </div>
 
               </p>
 
               </p>
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               </p>
 
               </p>
 
               <p>
 
               <p>
                   To demonstrate that MstX is beneficial to cell-free system not only for membrane protein expression, it was fused with single-chain variable fragment (scFv; Fig. 3). It was decided to do so as scFvs are usually prone to form aggregates and lose their function <var>in vitro</var>. We thought that MstX could stabilize scFvs and prevent aggregation. As a result, we observed that MstX fusion could enhance scFv solubility allowing it to use in cell-free systems.  
+
                   To demonstrate that MstX is beneficial to cell-free system not only for membrane protein expression, it was fused with single-chain variable fragment (scFv; Fig. 2). It was decided to do so as scFvs are usually prone to form aggregates and lose their function <var>in vitro</var>. We thought that MstX could stabilize scFvs and prevent aggregation. As a result, we observed that MstX fusion could enhance scFv solubility allowing it to use in cell-free systems.  
 
               </p>
 
               </p>
 
               <p>
 
               <p>
 
                     <div class="image-container">
 
                     <div class="image-container">
 
                             <img src="https://static.igem.org/mediawiki/2018/7/79/T--Vilnius-Lithuania--Fig4_Mistic.png"/>
 
                             <img src="https://static.igem.org/mediawiki/2018/7/79/T--Vilnius-Lithuania--Fig4_Mistic.png"/>
                             <p><strong>Fig. 4 </strong> Single-chain variable fragment (scFv) expression in IVTT system; SDS-PAGE. M - protein ladder, + - positive control DHFR, 1 - scFv, 2 - MstX-scFv, - negative control (without template DNA)</p>
+
                             <p><strong>Fig. 3 </strong> Single-chain variable fragment (scFv) expression in IVTT system; SDS-PAGE. M - protein ladder, + - positive control DHFR, 1 - scFv, 2 - MstX-scFv, - negative control (without template DNA)</p>
 
               </div>
 
               </div>
 
               <p>
 
               <p>
                   Analyzing the reaction samples, sediments in scFv were observed, which meant that scFv aggregated. However, in MstX-scFv sample there were no sediments. By analysing electrophoresis results (Fig. 4) it can be seen that MstX prevented formation of the aggregates which resulted in higher scFv expression yield.
+
                   Analyzing the reaction samples, sediments in scFv were observed, which meant that scFv aggregated. However, in MstX-scFv sample there were no sediments. By analysing electrophoresis results (Fig. 3) it can be seen that MstX prevented formation of the aggregates which resulted in higher scFv expression yield.
 
               </p>
 
               </p>
 
               <p></p>
 
               <p></p>
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               </p>
 
               </p>
 
               <p>scFv consists of a minimal functional antigen-binding domain of an antibody (~30 kDa) (Fig. 1) , in which the heavy variable chain (VH) and light variable chain (VL) are connected by Ser and Gly rich flexible linker. [1] In most cases scFv is expressed in bacteria, where it is produced in cytoplasm, a reducing environment, in which disulfide bonds are not able to form and protein is quickly degraded or aggregated. Although poor solubility and affinity limit scFvs’ applications, their stability can be improved by merging with other proteins. [2] When expressed in cell free system, scFv should form disulfide bonds with the help of additional molecules. Merging to a membrane protein would provide additional stability and would display scFv on liposome membrane, where its activity could be detected. These improved qualities make ScFv recombinant proteins a perfect tool to evaluate, if SynDrop system acts in an anticipated manner. Of all possible scFvs we decided to use scFv-anti vaginolysin, which binds and neutralizes toxin vaginolysin (VLY). Its main advantage is rapid (< 1 h) and cheap detection of activity by inhibition of erythrocyte lysis (Fig. 2). Looking into future applications, scFvs are also attractive targets of molecular evolution, because one round of  evolution would last less than one day thus generating a and wide range of different scFv mutants. Those displaying the highest affinity for antigens could be selected and used as drugs or drug carriers. </p>
 
               <p>scFv consists of a minimal functional antigen-binding domain of an antibody (~30 kDa) (Fig. 1) , in which the heavy variable chain (VH) and light variable chain (VL) are connected by Ser and Gly rich flexible linker. [1] In most cases scFv is expressed in bacteria, where it is produced in cytoplasm, a reducing environment, in which disulfide bonds are not able to form and protein is quickly degraded or aggregated. Although poor solubility and affinity limit scFvs’ applications, their stability can be improved by merging with other proteins. [2] When expressed in cell free system, scFv should form disulfide bonds with the help of additional molecules. Merging to a membrane protein would provide additional stability and would display scFv on liposome membrane, where its activity could be detected. These improved qualities make ScFv recombinant proteins a perfect tool to evaluate, if SynDrop system acts in an anticipated manner. Of all possible scFvs we decided to use scFv-anti vaginolysin, which binds and neutralizes toxin vaginolysin (VLY). Its main advantage is rapid (< 1 h) and cheap detection of activity by inhibition of erythrocyte lysis (Fig. 2). Looking into future applications, scFvs are also attractive targets of molecular evolution, because one round of  evolution would last less than one day thus generating a and wide range of different scFv mutants. Those displaying the highest affinity for antigens could be selected and used as drugs or drug carriers. </p>
               <img src="https://static.igem.org/mediawiki/2018/9/99/T--Vilnius-Lithuania--_Fig1_Surface-scFv.png"
+
              <div class="image-container">
 +
               <img src="https://static.igem.org/mediawiki/2018/1/16/T--Vilnius-Lithuania--PERMATOMAS_ScFv.png"/>
 
               <p><strong>Fig. 1 </strong>Simplified structure of scFv Antibody</p>
 
               <p><strong>Fig. 1 </strong>Simplified structure of scFv Antibody</p>
               <img src="https://static.igem.org/mediawiki/2018/9/97/T--Vilnius-Lithuania--Fig2_NEW_real_Surface_scFV.png"
+
            </div>
 +
            <div class="image-container">
 +
               <img src="https://static.igem.org/mediawiki/2018/9/97/T--Vilnius-Lithuania--Fig2_NEW_real_Surface_scFV.png"/>
 
               <p><strong>Fig. 2 </strong>Scheme of scFv_antiVLY and VLY interaction. Left- scFv_antiVLY binds to VLY, erythrocytes stay intact, Right- scFv_antiVLY does not bind and VLY lyse erythrocytes.</p>
 
               <p><strong>Fig. 2 </strong>Scheme of scFv_antiVLY and VLY interaction. Left- scFv_antiVLY binds to VLY, erythrocytes stay intact, Right- scFv_antiVLY does not bind and VLY lyse erythrocytes.</p>
 +
            </div></p>
 
               <p></p>
 
               <p></p>
 
               <h1>Results</h1>
 
               <h1>Results</h1>
 
               <p></p>
 
               <p></p>
               <p>scFv constructs were created <a href="http://parts.igem.org/Part:BBa_K2622004"> BBa_K2622004. and checked by <a href="https://2018.igem.org/Team:Vilnius-Lithuania/Protocols"> colony PCR and DNA sequencing. scFv synthesis was performed in a cell free system. Validation of protein expression was done by running a sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), see (Fig. 3)</p>
+
              <div class="image-container"></div>
               <img src="https://static.igem.org/mediawiki/2018/8/83/T--Vilnius-Lithuania--_Fig2_Surface-scFv.png"
+
               <p>scFv constructs were created <a href="http://parts.igem.org/Part:BBa_K2622004"> BBa_K2622004</a>. and checked by <a href="https://2018.igem.org/Team:Vilnius-Lithuania/Protocols"> colony PCR and DNA sequencing</a>. scFv synthesis was performed in a cell free system. Validation of protein expression was done by running a sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), see (Fig. 3)</p>
 +
               <img src="https://static.igem.org/mediawiki/2018/8/83/T--Vilnius-Lithuania--_Fig2_Surface-scFv.png">
 +
               
 
               <p><strong>Fig. 3 </strong> SDS-PAGE of scFv. GFP is used as positive control, C- chaperone DnaK.</p>
 
               <p><strong>Fig. 3 </strong> SDS-PAGE of scFv. GFP is used as positive control, C- chaperone DnaK.</p>
 +
            </div>
 +
             
 
               <p>Red arrows in the photo indicate scFv anti-vaginolysin (~27 kDa). As successful synthesis was confirmed, the next step was to check if protein folded correctly and was able to bind its antigen -  vaginolysin. We examined this by erythrocyte-lysis test, which was performed by comparing erythrocytes incubated with VLY (erythrocytes burst open) and erythrocytes incubated with VLY that was previously incubated with scFv anti-vaginolysin (less or no erythrocyte lysis). Results revealed that scFv binded to vaginolysin and inhibited cell lysis. Graph in (Fig. 4) demonstrates that scFv indeed attenuated the lysis of erythrocytes. These result prove scFv activity in IVTT system.</p>
 
               <p>Red arrows in the photo indicate scFv anti-vaginolysin (~27 kDa). As successful synthesis was confirmed, the next step was to check if protein folded correctly and was able to bind its antigen -  vaginolysin. We examined this by erythrocyte-lysis test, which was performed by comparing erythrocytes incubated with VLY (erythrocytes burst open) and erythrocytes incubated with VLY that was previously incubated with scFv anti-vaginolysin (less or no erythrocyte lysis). Results revealed that scFv binded to vaginolysin and inhibited cell lysis. Graph in (Fig. 4) demonstrates that scFv indeed attenuated the lysis of erythrocytes. These result prove scFv activity in IVTT system.</p>
               <img src="https://static.igem.org/mediawiki/2018/7/7b/T--Vilnius-Lithuania--_Fig3_Surface-scFv.png"
+
<div class="image-container">
 +
               <img src="https://static.igem.org/mediawiki/2018/7/7b/T--Vilnius-Lithuania--_Fig3_Surface-scFv.png">
 +
              <p>
 
               <p><strong>Fig. 4 </strong> Percentage of erythrocyte lysis at different +/-scFv dilutions.</p>
 
               <p><strong>Fig. 4 </strong> Percentage of erythrocyte lysis at different +/-scFv dilutions.</p>
              <p>We then went one step further and constructed MstX-scFv_antiVLY <a href="http://parts.igem.org/Part:BBa_K2622038"> BBa_2622038, fusion protein, aiming to increase the stability of scFv having in mind future applications and experiments of exposing it on liposome surface. Fusion protein was expressed in E.coli cells; yellow to red arrows in (Fig. 5A) indicate MstX-scFv expression after induction with IPTG.</p>
+
</div>             
 +
<p>We then went one step further and constructed MstX-scFv_antiVLY <a href="http://parts.igem.org/Part:BBa_K2622038"> BBa_2622038</a>, fusion protein, aiming to increase the stability of scFv having in mind future applications and experiments of exposing it on liposome surface. Fusion protein was expressed in E.coli cells; yellow to red arrows in (Fig. 5A) indicate MstX-scFv expression after induction with IPTG.</p>
 
               <p>Finally, we expressed the protein in a cell free system (Fig. 5B) along with scFv in order to compare how well scFv accomplishes its function alone or binded to other protein. In this case MstX-scFv_antiVLY fusion did not show superior activity than scFv_antiVLY alone (Fig. 6). These results also reveal that scFv_antiVLY is very sensitive and loses its activity with time. Ist and IInd attempts were separated by 1-2 hours. This amount of time is enough to measure decreasing activity. This must be taken into account when performing future experiments.</p>
 
               <p>Finally, we expressed the protein in a cell free system (Fig. 5B) along with scFv in order to compare how well scFv accomplishes its function alone or binded to other protein. In this case MstX-scFv_antiVLY fusion did not show superior activity than scFv_antiVLY alone (Fig. 6). These results also reveal that scFv_antiVLY is very sensitive and loses its activity with time. Ist and IInd attempts were separated by 1-2 hours. This amount of time is enough to measure decreasing activity. This must be taken into account when performing future experiments.</p>
               <img src="https://static.igem.org/mediawiki/2018/9/9c/T--Vilnius-Lithuania--Fig_4._5._Surface_scFv.png"
+
               <img src="https://static.igem.org/mediawiki/2018/9/9c/T--Vilnius-Lithuania--Fig_4._5._Surface_scFv.png">
 +
              <p>
 
               <p><strong>Fig. 5 </strong>A- MstX-scFv_antiVLY expression in Escherichia coli. B- scFv_antiVLY and MstX-scFv_antiVLY expression in cell-free system.</p>
 
               <p><strong>Fig. 5 </strong>A- MstX-scFv_antiVLY expression in Escherichia coli. B- scFv_antiVLY and MstX-scFv_antiVLY expression in cell-free system.</p>
               <img src="https://static.igem.org/mediawiki/2018/0/0c/T--Vilnius-Lithuania--_Fig6_Surface-scFv.png"
+
               <img src="https://static.igem.org/mediawiki/2018/0/0c/T--Vilnius-Lithuania--_Fig6_Surface-scFv.png">             
               <p><strong>Fig. 6 </strong>Fig 6. Percentage of erythrocyte lysis at different scFv/MstX-scFv dilutions.</p>
+
              <p>
 +
               <p><strong>Fig. 6 </strong> Percentage of erythrocyte lysis at different scFv/MstX-scFv dilutions.</p>
 
               <h1>Conclusions</h1>
 
               <h1>Conclusions</h1>
 
               <p>
 
               <p>
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               <p>Small size of scFv makes it a widely researched antibody. It’s ability to penetrate deeply into tissues and trait to elicit low to none organism’s immune response, makes scFv the one of the best candidates for medical, diagnostic, and research applications [3]. Efficient and fast method for scFv generation is in demand. SynDrop liposome display system offers an ability to produce scFv in IVTT system and display them on membranes to facilitate rapid antigen binding. scFv on the other hand can also help us prove that our system work either cost efficiently or with extreme precision. scFv surface display is compatible with using fluorescence assisted cell sorting (FACS) to detect well functioning liposomes. [4] This would reduce amount of time needed for mutant sorting compared with enzyme-linked immunosorbent assay. Second, scFv display is compatible with the experiments described in this section. We have performed erythrocyte-lysis tests to prove the functional activity of scFv anti-vaginolysin that was synthesized in the IVTT system. Not all experiments with VLY indicated positive antibody activity. We hypothesized that proteins could have aggregated very quickly after IVTT expression and the amount of active antibody left in the solution was not enough to inhibit VLY in quantities, what would have been detectable. This hypothesis was further supported by several experiments, which revealed decreasing scFv antibody’s functional activity with time. Moreover, not every experiment was done just after IVTT reaction completed and spend few hours in +4 ˚C. Another option to test scFv, single or displayed on liposomes to gain most reliable results, is an ELISA test. It requires specific antibodies and tags (His-6x or Strep-tag) on scFv or MstX-scFv. </p>
 
               <p>Small size of scFv makes it a widely researched antibody. It’s ability to penetrate deeply into tissues and trait to elicit low to none organism’s immune response, makes scFv the one of the best candidates for medical, diagnostic, and research applications [3]. Efficient and fast method for scFv generation is in demand. SynDrop liposome display system offers an ability to produce scFv in IVTT system and display them on membranes to facilitate rapid antigen binding. scFv on the other hand can also help us prove that our system work either cost efficiently or with extreme precision. scFv surface display is compatible with using fluorescence assisted cell sorting (FACS) to detect well functioning liposomes. [4] This would reduce amount of time needed for mutant sorting compared with enzyme-linked immunosorbent assay. Second, scFv display is compatible with the experiments described in this section. We have performed erythrocyte-lysis tests to prove the functional activity of scFv anti-vaginolysin that was synthesized in the IVTT system. Not all experiments with VLY indicated positive antibody activity. We hypothesized that proteins could have aggregated very quickly after IVTT expression and the amount of active antibody left in the solution was not enough to inhibit VLY in quantities, what would have been detectable. This hypothesis was further supported by several experiments, which revealed decreasing scFv antibody’s functional activity with time. Moreover, not every experiment was done just after IVTT reaction completed and spend few hours in +4 ˚C. Another option to test scFv, single or displayed on liposomes to gain most reliable results, is an ELISA test. It requires specific antibodies and tags (His-6x or Strep-tag) on scFv or MstX-scFv. </p>
 
               </p>
 
               </p>
               <h2>Refferences</h2>
+
               <h2>References</h2>
 
               <p>
 
               <p>
 
               <ol>
 
               <ol>
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         </section>
 
         </section>
 
       </div>
 
       </div>
  </div>
 
 
   </div>
 
   </div>
 
   <div class="carrot-back">
 
   <div class="carrot-back">

Latest revision as of 20:11, 30 November 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.

invert