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

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                   <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.
 
                   <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.
 
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                   <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.
 
                   <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.
 
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                   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.

Revision as of 23:51, 17 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.

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