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

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               <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>
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               <img src="https://static.igem.org/mediawiki/2018/7/7b/T--Vilnius-Lithuania--_Fig3_Surface-scFv.png"
 
               <img src="https://static.igem.org/mediawiki/2018/7/7b/T--Vilnius-Lithuania--_Fig3_Surface-scFv.png"
 
                
 
                
 
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               <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</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>
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<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"

Revision as of 03:10, 18 October 2018

Design and Results

Results

Cell-free, synthetic biology systems open new horizons in engineering biomolecular systems which feature complex, cell-like behaviors in the absence of living entities. Having no superior genetic control, user-controllable mechanisms to regulate gene expression are necessary to successfully operate these systems. We have created a small collection of synthetic RNA thermometers that enable temperature-dependent translation of membrane proteins, work well in cells and display great potential to be transferred to any in vitro protein synthesis system.

invert