Difference between revisions of "Team:Washington/Aptamer"

 
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                             <a href="https://2015.igem.org/Team:Washington/Paper_Device#Methods">Methods</a>
 
                             <a href="https://2015.igem.org/Team:Washington/Paper_Device#Methods">Methods</a>
 
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                                <a href="https://2015.igem.org/Team:Washington/Design">Design</a>
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                                 <a href="https://2015.igem.org/Team:Washington/Paper_Device#Results">Results</a>
 
                                 <a href="https://2015.igem.org/Team:Washington/Paper_Device#Results">Results</a>
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                             <a href="https://2015.igem.org/Team:Washington/Team#Attributions">Attributions</a>
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                             <a href="https://2015.igem.org/Team:Washington/Attributions">Attributions</a>
 
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        <h2> Heading </h2>
 
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         <h2>
 
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             <div id="Experiments">Aptamer Background</div>
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             <div id="Introduction">Aptazyme Background</div>
 
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        <p>For commercial shellfish farmers and recreational hunters alike, marine biotoxins pose a significant threat to health and welfare. With this project, we aim to create an inexpensive and easy-to-use test kit for the detection of the shellfish toxin okadaic acid using engineered yeast strains and DNA aptamers on a paper device. We also hope that this project paves the way for a new class of biosensors capable of detecting a wide range of small molecules. </p>
 
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        <p>An aptamer is a single strand of RNA which folds into a structure that is able to bind to a variety of small molecules and proteins. Theophylline is commonly used target molecule for academic studies on aptamers due to its ability to permeate membranes. </p>
 
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        <p>Ribozymes are self-cleaving pieces of RNA. This can be utilized to destabilize RNA transcripts.</p>
 
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        <p>Aptazymes are a combination of both aptamers and ribozymes. This allows for the selectivity of aptamers which in turn results in the activity of the ribozyme section of the RNA. Overall this is a reactive strand of RNA with selectivity. Additionally, this can control protein expression at the level of translation. This allows for quicker response times.
 
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RNA allows for easy tenability so that the general system can be modified to fit several contexts. In this project, we use theophiline as a model small molecule due to its ability to permeate through membranes.</p>
 
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        <h2>Aptazyme Protocols:</h2>
 
  
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      <p align = "justify"> An aptamer is a single strand of RNA which folds into a structure that is able to bind to a variety of small molecules and proteins. Ribozymes are in some contexts self-cleaving pieces of RNA, which can be utilized to destabilize RNA transcripts. A specific subgroup of Aptazymes, called Riboswitches, are a combination of both aptamers and ribozymes. Putting these components together allows for the aptamer section of the RNA to regulate the activity of the ribozyme section of the RNA. Overall, riboswitch is a reactive strand of RNA under allosteric control. Additionally, aptazymes can control protein expression at the level of translation. This allows for quicker response times compared to the traditional method of modifying rates of transcription through interactions with promoters. Theophylline is commonly used target molecule (ligand for the aptazyme) for academic studies on aptamers due to its ability to permeate membranes.</p>
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      <li><a href="#YCI">Yeast Chromosome Integration</a></li>
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        <h2>
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      <li><a href="#TS">Theophylline Stock</a></li>
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            <div id="Methods"> Design and Methods</div>
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      <li><a href="#F_R">Fluorescence Reading</a></li>
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        </h2>
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    </ul>
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    <div class="tabContent" id="YCI">
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<p>Since we thought yeast would be a better implementation on paper, our original concept was to utilize an aptazyme sequence that was contributed by the Smolke lab that was found to be highly functional in yeast. This sequence was integrated with Venus, an optimized form of a yellow fluorescent protein (YFP); a yeast GPD Promoter; a cyc1 terminator; and a uracil auxotrophic marker. The plasmid was cloned in e. coli and digested with a PmeI restriction enzyme in order to insert the gene into the yeast genome via homologous integration by the uracil casette. Experiments were carried out using these new strains of yeast. </p>
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      <div>
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<ul style="list-style-type:circle">
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        <h2>
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      <li>Digest E. Coli plasmid using PmeI restriction enzyme</li>
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            <div id="Results"> Results </div>
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<li>Digest E. Coli plasmid using PmeI restriction enzyme<li>
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        </h2>
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<li>1 ug of DNA<li>
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<li>5 uL of 10x NEB CutSmart buffer<li>
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<p>Our initial results from the experiment with the theophylline induced and non-induced conditions showed a systematic difference with all induced trials having a higher mean fluorescence/cell (graph). yeast transformant 3 showed a greater response than the other transformants.</p>
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<li>1 uL of restriction enzyme<li>
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<li>Fill to 50 uL with water<li>
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<p>Our subsequent concentration dependence experiment showed a increase in fluorescence with increasing concentrations of theophylline. Here again the transformant 3 showed the best concentration dependence. (graph)</p>
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<li>Incubate at 37 C for 15 minutes (1 hour if not using TimeSaver buffer)<li>
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<li>Heat inactivate at 65 C for 20 minutes<li>
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<p>The paper device/Gal promoter tests showed fluorescence in all galactose present conditions. The difference in fluorescence between the caffeine and theophylline conditions was very hard to spot, though we expected the caffeine condition to show lesser fluorescence. The device which was plated on sugar free media and had galactose injected prior to theophylline induction also showed fluorescence. (picture)</p>
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<li>Agarose gel purify (optional)<li>
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<li>Salmon Sperm Transformation<li>
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<li>Grow a yeast overnight <li>
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        <h2>
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<li>Check OD of culture. 0.5-0.6 are the preferred readings, if the reading is lower, wait for longer growth, if the reading is higher, dilute the sample.<li>
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            <div id="Conclusion"> Conclusion </div>
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<li>Spin down 10 ml of cells per transformation.<li>
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        </h2>
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<li>Decant supernatant and wash with 10 ml ddH2O. Vortex to resuspend and spin down.<li>
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<li>Remove the supernatant.<li>
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<p align = "justify"> Contents </p>
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<li>Resuspend cells in 300 uL .1 M LiOAc. Transfer to a 1.5 mL tube.<li>
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<li>Incubate at 30 C for 15 min<li>
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        <h2>
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<li>Put salmon sperm DNA in boiling water for 5 minutes. Cool immediately on ice.<li>
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            <div id="Future_Direction"> Future Directions </div>
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<li>Spin down cells and remove supernatant.<li>
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        </h2>
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<li>Add the following in order:<li>
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<li>240 uL 50% PEG<li>
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<p align = "justify"> Given that the aptazyme is a model system instead of actually selecting for specific toxins, the next step would be to create aptamers that select for the toxins of interest. Most likely, the selection process would follow evolution based methods (Goler et al., 2014) to optimize a sequence of RNA that would react with the selected molecules. However, this lengthy process does not guarantee results in a set timeframe which is why this portion of the project was not undertaken over the summer.
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<li>36 uL 1.0 M LioAc<li>
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There are multiple methods to improve the user friendliness. One route to take would be to lower the basal expression levels or increase the expression levels of YFP when the theophylline/toxin is present. A method we are undertaking that will not produce results in time for the wiki is the inclusion of additional aptazyme copies on the RNA (Wei et al, 2013; Win and Smolke, 2008) which should decrease the basal level of expression resulting in a greater proportion between active and basal expression.  
−
<li>10 uL salmon sperm DNA<li>
+
Another method to improve user friendliness would be to create more instantaneous colorimetric switches. A classic example is the creation of RNA-based cocaine sensors (Stojanovic et al., 2001). These sensors should provide feedback to the presence of toxins at a much faster rate than the aptazyme system in yeast. Additionally, the development of better colorimetric and fluorescent probes such as Spinach allow for more robust responses in these systems.   </p>
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<li>34 uL DNA<li>
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<li>40 uL ddH2O<li>
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        <h2>
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<li>Final volume: 360 uL<li>
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            <div id="Biobrick"> Biobricks </div>
 +
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      </div>
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<p align = "justify"> Contents</p>
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    </div>
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<div class="tabContent" id="TS">
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      <div>
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<p>The auxin pathway produces a color output rather than a fluorescent one….(I will finish this part tomorrow when my computer isn’t dead)
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Microfluidic design:.....(1% remaining….)</p>
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      </div>
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    </div>
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<div class="tabContent" id="F_R">
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−
      <div>
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<p>In this design RNA aptamers are used to sense our target molecule, theophylline.  Aptamers, unlike antibodies, can actually bind to virtually any molecule, allowing for a more versatile system.  We’ve implemented a ribozyme switch which, when active, cleaves the mRNA code of our target sequence, hindering the production of GFP by default.  However, in the presence of theophylline our switch becomes inactive, allowing for the expression of our target gene.  This system is useful because it is faster-acting than more traditional expression pathways, and can be generalized to many other small molecules by changing the aptamer sequence.</p2>
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          <div>Design</div>
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Latest revision as of 03:12, 19 September 2015



Aptazyme Background

An aptamer is a single strand of RNA which folds into a structure that is able to bind to a variety of small molecules and proteins. Ribozymes are in some contexts self-cleaving pieces of RNA, which can be utilized to destabilize RNA transcripts. A specific subgroup of Aptazymes, called Riboswitches, are a combination of both aptamers and ribozymes. Putting these components together allows for the aptamer section of the RNA to regulate the activity of the ribozyme section of the RNA. Overall, riboswitch is a reactive strand of RNA under allosteric control. Additionally, aptazymes can control protein expression at the level of translation. This allows for quicker response times compared to the traditional method of modifying rates of transcription through interactions with promoters. Theophylline is commonly used target molecule (ligand for the aptazyme) for academic studies on aptamers due to its ability to permeate membranes.

Design and Methods

Since we thought yeast would be a better implementation on paper, our original concept was to utilize an aptazyme sequence that was contributed by the Smolke lab that was found to be highly functional in yeast. This sequence was integrated with Venus, an optimized form of a yellow fluorescent protein (YFP); a yeast GPD Promoter; a cyc1 terminator; and a uracil auxotrophic marker. The plasmid was cloned in e. coli and digested with a PmeI restriction enzyme in order to insert the gene into the yeast genome via homologous integration by the uracil casette. Experiments were carried out using these new strains of yeast.

Results

Our initial results from the experiment with the theophylline induced and non-induced conditions showed a systematic difference with all induced trials having a higher mean fluorescence/cell (graph). yeast transformant 3 showed a greater response than the other transformants.

Our subsequent concentration dependence experiment showed a increase in fluorescence with increasing concentrations of theophylline. Here again the transformant 3 showed the best concentration dependence. (graph)

The paper device/Gal promoter tests showed fluorescence in all galactose present conditions. The difference in fluorescence between the caffeine and theophylline conditions was very hard to spot, though we expected the caffeine condition to show lesser fluorescence. The device which was plated on sugar free media and had galactose injected prior to theophylline induction also showed fluorescence. (picture)

Conclusion

Contents

Future Directions

Given that the aptazyme is a model system instead of actually selecting for specific toxins, the next step would be to create aptamers that select for the toxins of interest. Most likely, the selection process would follow evolution based methods (Goler et al., 2014) to optimize a sequence of RNA that would react with the selected molecules. However, this lengthy process does not guarantee results in a set timeframe which is why this portion of the project was not undertaken over the summer. There are multiple methods to improve the user friendliness. One route to take would be to lower the basal expression levels or increase the expression levels of YFP when the theophylline/toxin is present. A method we are undertaking that will not produce results in time for the wiki is the inclusion of additional aptazyme copies on the RNA (Wei et al, 2013; Win and Smolke, 2008) which should decrease the basal level of expression resulting in a greater proportion between active and basal expression. Another method to improve user friendliness would be to create more instantaneous colorimetric switches. A classic example is the creation of RNA-based cocaine sensors (Stojanovic et al., 2001). These sensors should provide feedback to the presence of toxins at a much faster rate than the aptazyme system in yeast. Additionally, the development of better colorimetric and fluorescent probes such as Spinach allow for more robust responses in these systems.

Biobricks

Contents