Difference between revisions of "Team:Kent/HP/Silver"

 
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                     <ul class="drop-menu menu-1">
 
                     <ul class="drop-menu menu-1">
 
                         <a href="https://2017.igem.org/Team:Kent/Description"><li>Description</li></a>
 
                         <a href="https://2017.igem.org/Team:Kent/Description"><li>Description</li></a>
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<a href="https://2017.igem.org/Team:Kent/Design"><li> Design </li></a>
 
 
                       <a href="https://2017.igem.org/Team:Kent/Results"><li>Results</li></a>
 
                       <a href="https://2017.igem.org/Team:Kent/Results"><li>Results</li></a>
 
                         <a href="https://2017.igem.org/Team:Kent/Model"><li>Modelling</li></a>
 
                         <a href="https://2017.igem.org/Team:Kent/Model"><li>Modelling</li></a>
−
<a href="https://2017.igem.org/Team:Kent/Demonstrate"><li>Demonstrate</li></a>
+
 
 
                     </ul>
 
                     </ul>
 
                 <li>
 
                 <li>
 
                     <a href="#">Parts</a>
 
                     <a href="#">Parts</a>
 
                     <ul class="drop-menu menu-2">
 
                     <ul class="drop-menu menu-2">
−
<a href="https://2017.igem.org/Team:Kent/Parts"> <li> Parts </li></a>
 
 
                         <a href="https://2017.igem.org/Team:Kent/Basic_Part"><li>Basic Parts</li></a>
 
                         <a href="https://2017.igem.org/Team:Kent/Basic_Part"><li>Basic Parts</li></a>
−
                        <a href="https://2017.igem.org/Team:Kent/Composite_Part"><li>Composite Parts</li></a>
+
 
−
<a href = "https://2017.igem.org/Team:Kent/Part_Collection"><li> Part Collection </li></a>
+
  
 
                     </ul>
 
                     </ul>
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                     <a href="#">Human Practices</a>
 
                     <a href="#">Human Practices</a>
 
                     <ul class="drop-menu menu-2">
 
                     <ul class="drop-menu menu-2">
−
                         <a href="https://2017.igem.org/Team:Kent/HP/Silver"><li>Silver</li></a>
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                         <a href="https://2017.igem.org/Team:Kent/HP/Silver"><li>Integrated HP</li></a>
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<a href="https://2017.igem.org/Team:Kent/HP/Gold_Integrated"><li>Gold</li></a>
+
 
                         <a href="https://2017.igem.org/Team:Kent/Engagement"><li>Public Engagement</li></a>
 
                         <a href="https://2017.igem.org/Team:Kent/Engagement"><li>Public Engagement</li></a>
 
                         <a href="https://2017.igem.org/Team:Kent/InterLab"><li>Interlab</li></a>
 
                         <a href="https://2017.igem.org/Team:Kent/InterLab"><li>Interlab</li></a>
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         <div id ="title">
 
         <div id ="title">
 
                 <img src = "https://static.igem.org/mediawiki/2017/thumb/4/46/T--Kent--SilverHeader.png/401px-T--Kent--SilverHeader.png" id="header1">  
 
                 <img src = "https://static.igem.org/mediawiki/2017/thumb/4/46/T--Kent--SilverHeader.png/401px-T--Kent--SilverHeader.png" id="header1">  
−
<span>Silver</span>
+
<span>Integrated HP</span>
 
<img src = "https://static.igem.org/mediawiki/2017/thumb/1/12/T--Kent--SilverHeader2.png/766px-T--Kent--SilverHeader2.png" id="header2">
 
<img src = "https://static.igem.org/mediawiki/2017/thumb/1/12/T--Kent--SilverHeader2.png/766px-T--Kent--SilverHeader2.png" id="header2">
 
         </div>
 
         </div>
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<p>Our team has strived to make human practices the solid foundation of our project right from the start. We wanted to ensure we fully understood the implications of any project we chose to pursue while addressing current global problems.
 +
<br>
 +
We held a brainstorming session where we bounced ideas off of one another for potential project ideas and had narrowed it down to a top five. We decided we needed to consult with our immediate community to scope out what issues they deemed important that needed immediate addressing. Through different outreach opportunities, we were able to comprehend the controversy surrounding GMOs and genetic engineering in general. This caused us to critically analyze our project ideas before deciding upon ‘LuCAS’: a novel way of mRNA localization using CRISPR - dCas13a.
 +
</p>
 +
</div>
 +
</div>
 +
 
 +
<div class="centerizer"  id="title2">
 +
<span>Why CRISPR-dCas13a (C2C2)?</span>
 +
<div class="SlineSeparator"></div>
 +
</div>
 +
 
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<div id="box2">
 +
 
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<div id="textbox2" >
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<p>We were introduced to CRISPR-Cas9 as a tool for targeting cellular mRNA translation through Dr. Peter Ellis, a lecturer in Molecular Biology and Reproduction. However, through research, we discovered that Cas9 is a DNA endonuclease, meaning it recognizes DNA targets and cuts them. It would need to be ‘tricked’ by molecular means. C2C2, however, or Cas13a is an RNA endonuclease, meaning it cuts the RNA rather than the DNA. There are two key differences when comparing Cas13 to CAS9: Firstly, Cas13 recognizes RNA rather than DNA. Secondly, once the target is recognized, it acts promiscuously and starts to ‘chew up’ all of the RNA around it, not only the intended target sequence. <br><img src="https://static.igem.org/mediawiki/2017/3/3c/T--Kent--LogbookP5.png"><img src="https://static.igem.org/mediawiki/2017/5/52/T--Kent--LogbookP3.png">
 +
</p>
 +
</div>
 +
<div class="centerizer">
 +
<div class="connector">
 +
<img src="https://static.igem.org/mediawiki/2017/thumb/f/f4/T--Kent--HP9.png/133px-T--Kent--HP9.png">
 +
</div>
 +
<div id="textbox2" >
 +
<p>
 +
We wanted to utilize this knowledge and build upon the CRISPR-Cas9 and produce a tool that would just track the mRNA, not cut it. We did not want to overcomplicate our methodology and deemed tagging the mRNA with GFP would be sufficient as we would be able to essentially track the mRNA movement to ensure its localization without damaging its integrity through cutting it.
 +
</p>
 +
</div>
 +
</div>
 +
<div class="centerizer">
 +
<div class="connector">
 +
<img src="https://static.igem.org/mediawiki/2017/thumb/f/f4/T--Kent--HP9.png/133px-T--Kent--HP9.png">
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 +
</div>
 +
<nav class="droptext arrows">
 
<header class="hull">
 
<header class="hull">
−
<label for="acc-close" class="hull-title">Basic Protocols</label>
+
<label for="acc-close" class="hull-title">Surveys and Consultations</label>
 
</header>
 
</header>
 
<input type="radio" name="droptext" id="cb1" />
 
<input type="radio" name="droptext" id="cb1" />
 
<section class="hull">
 
<section class="hull">
−
<label class="hull-title" for="cb1">Production of Lysogeny broth (LB)</label>
+
<label class="hull-title" for="cb1">Surveys</label>
 
<label class="hull-close" for="acc-close"></label>
 
<label class="hull-close" for="acc-close"></label>
−
<div class="hull-content">For 1 litre of LB a mixture of 10g of sodium chloride, 10g peptone, 5g of yeast extract as well as 1
+
<div class="hull-content"><ul><li>We wanted the opportunity to receive insight from the public immediately, even before deciding on a project idea. We wanted to ensure that any topic we would be putting our focus into would heed positive results, not only from the scientific community but from the general public as well.</li>
−
litre of distilled water in a glass bottle. We then used a magnetic spinner to help mix the powders
+
 
−
with the water, we avoided shaking the glass bottle as it would cause froth and waste some of the
+
<li>We set out to design several surveys, questioning the community at our University and locally on their knowledge of GMOs and whether our project abstract was an idea they agreed would be beneficial once produced. </li>
−
LB.
+
 
−
<br>
+
<li>The general consensus from the students at the University was that they agreed genetic modification would be beneficial when used appropriately, however strict regulations would need to be in place when dealing with GMO applications, a small percentage even saying they would go the extra mile to ensure they did not deal with GMOs in their day to day lives.</li>
−
When making the LB we also made another litre batch and added 15g of agar extract to be able to
+
<li><img src="https://static.igem.org/mediawiki/2017/e/e8/T--Kent--HP1.png"></li>
−
grow bacteria on plates.</div>
+
<li>With this in mind, we introduced our project abstract and explained the positive outcomes we saw it producing and its potential as a diagnostic tool. The majority of the feedback we received was positive and allowed us to confidently advance in our laboratory work with a goal in mind: produce a tool with a promising future in diagnositcs.
 +
</li>
 +
<li><img src="https://static.igem.org/mediawiki/2017/d/d9/T--Kent--HP2.png"><img src="https://static.igem.org/mediawiki/2017/6/60/T--Kent--HP3.png"></li>
 +
<li><img src="https://static.igem.org/mediawiki/2017/8/84/T--Kent--HP4.png"></li>
 +
</div>
 
</section>
 
</section>
 +
<input type="radio" name="droptext" id="acc-close" />
 
<input type="radio" name="droptext" id="cb2" />
 
<input type="radio" name="droptext" id="cb2" />
 
<section class="hull">
 
<section class="hull">
−
<label class="hull-title" for="cb2">Production of SOB medium and magnesium stock</label>
+
<label class="hull-title" for="cb2">Professor Michelle Garrett</label>
 
<label class="hull-close" for="acc-close"></label>
 
<label class="hull-close" for="acc-close"></label>
−
<div class="hull-content">Bringing together 20g of tryptone, 5g of yeast extract, 0.584g of NaCl, 0.186g of KCl and mixing it
+
<div class="hull-content">When conceptualizing our project, we realized we would need to transfect our cell lines into mammalian cells if we wanted to have any use of it as a potential diagnostic tool, as it would be relatively useless in its bacterial cell host. Professor Garrett was able to guide us in the direction of the Smales Group at our University, providing us with the resources needed to make the transfections possible.<br><img src="https://static.igem.org/mediawiki/2017/2/2b/T--Kent--HP8.png"></div>
−
with 990ml of millipure water (using the magnetic mixer again) which was then put in to autoclave
+
−
to sterilise it, after it was taken out and let for it to cool down to below 60 o C.
+
−
<br>
+
−
10ml of 2M Mg 2+ stock was then added and then brought to 100ml with millipure water, 0.2mm
+
−
filter sterilize was then used</div>
+
 
</section>
 
</section>
 +
<input type="radio" name="droptext" id="acc-close" />
 
<input type="radio" name="droptext" id="cb3" />
 
<input type="radio" name="droptext" id="cb3" />
 
<section class="hull">
 
<section class="hull">
−
<label class="hull-title" for="cb3">Production of SOC medium and glucose stock</label>
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<label class="hull-title" for="cb3">Ian Brown, Microscopy Suite Facility Manager</label>
 
<label class="hull-close" for="acc-close"></label>
 
<label class="hull-close" for="acc-close"></label>
−
<div class="hull-content">Once again bring 20g of tryptone, 5g of yeast of extract, 0.584g of NaCl, 0.186g of KCL, and then
+
<div class="hull-content">As CRISPR itself has only recently been discovered as an mRNA tracking tool, most of the project entailed repeated trial and error. We realized before we even thought of imaging, we needed to ensure we had the appropriate controls for comparative and evaluative reasons. We were able to generate a repeatable layout for future imaging purposes that Ian Brown was able to confirm would be adequate in imaging our cells accurately<br>
−
bring 970 ml with millipure water and use the magnetic mixer once again, this was also then put in
+
<img src="https://static.igem.org/mediawiki/2017/9/9d/T--Kent--HP5.png"></div>
−
to autoclave.
+
−
<br>
+
−
10ml of 2M Mg 2+ stock and then bring it to 100ml with milllipure water, filter sterilize it with 0.2m
+
−
and then final add 20ml of 1M glucose stock.</div>
+
 
</section>
 
</section>
 
<input type="radio" name="droptext" id="acc-close" />
 
<input type="radio" name="droptext" id="acc-close" />
 
<input type="radio" name="droptext" id="cb4" />
 
<input type="radio" name="droptext" id="cb4" />
 
<section class="hull">
 
<section class="hull">
−
<label class="hull-title" for="cb4">Production of Glycerol stock</label>
+
<label class="hull-title" for="cb4">Dr. Emma Mead</label>
 
<label class="hull-close" for="acc-close"></label>
 
<label class="hull-close" for="acc-close"></label>
−
<div class="hull-content">If you wish to store bacteria long term, you will need to create a Glycerol Stock after
+
<div class="hull-content">Through research, we were able to conclude the best possible cells to transfect our constructs into would have to be HEK293 cells. However, without experience in such a field, we were guided to Dr. Mead, who proved to be invaluable throughout the entirety of our project. She confirmed that it would indeed have to be the HEK293 cells and provided us with samples to work with, as well as guiding us through the passaging and transfection itself.<br>
−
inoculating an overnight liquid culture
+
<img src="https://static.igem.org/mediawiki/2017/5/5a/T--Kent--HP6.png"></div>
−
<br>
+
−
<ul><li>Once bacterial growth has been achieved, 500μL of the overnight liquid
+
−
culture needs to be added to 500μL of 50% glycerol in a 2mL tube where it
+
−
should be gently mixed</li>
+
−
<li>The glycerol stock should then be frozen at -80 o C<ul>
+
−
<li> Successive freeze and thaw cycles will reduce the stocks shelf life</li></ul>
+
−
</li></ul></div>
+
 
</section>
 
</section>
−
<input type="radio" name="droptext" id="acc-close" />
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<input type="radio" name="droptext" id="acc-close" />
 
<input type="radio" name="droptext" id="cb5" />
 
<input type="radio" name="droptext" id="cb5" />
 
<section class="hull">
 
<section class="hull">
−
<label class="hull-title" for="cb5">Running Agarose Gel</label>
+
<label class="hull-title" for="cb5">Dr. Rosalyn Masterton</label>
 
<label class="hull-close" for="acc-close"></label>
 
<label class="hull-close" for="acc-close"></label>
−
<div class="hull-content">After the cells have been miniprepped and the plasmid put through a restriction digest, the agarose gel can be run.
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<div class="hull-content">We were able to conclude that we would need a vector to clone our construct before we could image it and decided upon pcDNA 3.1 (+) mammalian vector through the help of Dr. Masterton with her expertise in molecular biology. She also advised us who to speak to for the use of mammalian cell lines.<br>
−
<br>
+
<img src="https://static.igem.org/mediawiki/2017/b/bf/T--Kent--HP7.png"></div>
−
<ul><li>Make up some agarose. This is done by taking 0.5g of agarose powder and putting it in a
+
−
250ml sterile conical flask, with 50ml of TAE buffer, then microwaving it in small pulses (20
+
−
seconds then swirling it around) until it is dissolved. Don’t overheat it or it will evaporate too
+
−
much. Make up the evaporated volume to 50ml with distilled water.</li>
+
−
<li>Add 1 vial of cybersafe (ask technical services for a tube of it and add all of it)</li>
+
−
<li>Line the white sides of the tank with the agarose solution, to seal it and prevent leakage. Use
+
−
a p1000 pipette set to 1ml. Let it dry (about 5 mins max)</li>
+
−
<li>Then pour all the agarose/sybrsafe solution into the tank and put in the comb. Let it set and
+
−
solidify (maximum 30 mins)</li>
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−
<li>When the gel has set, remove the comb from the tank (gently!) and then cover the whole
+
−
tank with TAE buffer, so there’s at least half a centimetre of TAE covering the gel.</li>
+
−
<li>Now, the samples need to be loaded. Load some DNA markers (ask technical services for a
+
−
tube of this and load the whole tube) into well 1( left hand side) and then choose what you
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−
load into wells 2, 3, and 4 etc. (make sure you note what’s in each lane!)</li>
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<li>Load all of your digests into the wells 2,3, and 4.</li>
+
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<li>Plug into a power supply and put the cover on. Run for 40 mins to an hour at 80v. The amps
+
−
don’t matter.</li>
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−
<li>Once the visible markers have reached the half way point of the tank, turn off the power
+
−
supply and drain the TAE buffer form the tank. Remove the gel with a spatula and place in a
+
−
UV imaging box. Take an image of the gel under UV light, save it onto a USB stick.</li></ul></div>
+
 
</section>
 
</section>
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<input type="radio" name="droptext" id="acc-close" />
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Latest revision as of 03:03, 16 December 2017


Integrated HP

Our team has strived to make human practices the solid foundation of our project right from the start. We wanted to ensure we fully understood the implications of any project we chose to pursue while addressing current global problems.
We held a brainstorming session where we bounced ideas off of one another for potential project ideas and had narrowed it down to a top five. We decided we needed to consult with our immediate community to scope out what issues they deemed important that needed immediate addressing. Through different outreach opportunities, we were able to comprehend the controversy surrounding GMOs and genetic engineering in general. This caused us to critically analyze our project ideas before deciding upon ‘LuCAS’: a novel way of mRNA localization using CRISPR - dCas13a.

Why CRISPR-dCas13a (C2C2)?

We were introduced to CRISPR-Cas9 as a tool for targeting cellular mRNA translation through Dr. Peter Ellis, a lecturer in Molecular Biology and Reproduction. However, through research, we discovered that Cas9 is a DNA endonuclease, meaning it recognizes DNA targets and cuts them. It would need to be ‘tricked’ by molecular means. C2C2, however, or Cas13a is an RNA endonuclease, meaning it cuts the RNA rather than the DNA. There are two key differences when comparing Cas13 to CAS9: Firstly, Cas13 recognizes RNA rather than DNA. Secondly, once the target is recognized, it acts promiscuously and starts to ‘chew up’ all of the RNA around it, not only the intended target sequence.

We wanted to utilize this knowledge and build upon the CRISPR-Cas9 and produce a tool that would just track the mRNA, not cut it. We did not want to overcomplicate our methodology and deemed tagging the mRNA with GFP would be sufficient as we would be able to essentially track the mRNA movement to ensure its localization without damaging its integrity through cutting it.