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

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                             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.  
 
                             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.  
 
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                             <strong>Fig. 9 a</strong> brightfield image of the liposomes that contain IVTT system and plasmid GFP DNA (after incubation);
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                            <img src="https://static.igem.org/mediawiki/2018/c/c2/T--Vilnius-Lithuania--Fig9_Liposomes.png"/>
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                             <strong>Fig 9 </strong> brightfield image of the liposomes that contain IVTT system and plasmid GFP DNA (after incubation);
 
                             scale bar is 10 µm; <strong>b</strong> liposomes imaged with FITC: fluorescence confirms that transcription and translation
 
                             scale bar is 10 µm; <strong>b</strong> liposomes imaged with FITC: fluorescence confirms that transcription and translation
 
                             reactions occur inside them; scale bar is 10 µm; <strong>c</strong> liposomes containing purified GFP protein: all the
 
                             reactions occur inside them; scale bar is 10 µm; <strong>c</strong> liposomes containing purified GFP protein: all the
                             liposomes exhibit fluorescence validating excellent encapsulation efficiency; scale bar is 20 µm.                          
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                             liposomes exhibit fluorescence validating excellent encapsulation efficiency; scale bar is 20 µm.                                    
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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.  
 
                             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.  
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                            <strong>Fig. 10 a</strong> concentrated calcein encapsulated within liposomes: the outer solution fluoresces as some of the liposomes
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                            inevitably burst releasing calcein into the outside; <strong>b</strong> box plot comparison of the control (without α-hemolysin) and
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                                <img src="https://static.igem.org/mediawiki/2018/3/34/T--Vilnius-Lithuania--Fig10_Liposomes.png"/>
                            a group with inserted α-hemolysin; nonparametrical Mann-Whitney U test was used for the statistical evaluation:
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                                <strong>Fig 10 a</strong> concentrated calcein encapsulated within liposomes: the outer solution fluoresces as some of the liposomes
                            the group with α-hemolysin shows statistically significant (p < 0.0001) increase in fluorescence                          
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                                inevitably burst releasing calcein into the outside; <strong>b</strong> box plot comparison of the control (without α-hemolysin) and
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                                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                                                  
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Revision as of 00:27, 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.

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