Difference between revisions of "Team:Kent/Improve"

 
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#title span{
 
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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>
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<a href="https://2017.igem.org/Team:Kent/Model"><li>Modelling</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>
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                         <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>
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                     </ul>
 
                     </ul>
 
                 <li>
 
                 <li>
 
                     <a href="#">Parts</a>
 
                     <a href="#">Parts</a>
 
                     <ul class="drop-menu menu-2">
 
                     <ul class="drop-menu menu-2">
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<a href="https://2017.igem.org/Team:Kent/Parts"> <li> Parts </li></a>
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                         <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>
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                         <a href="https://2017.igem.org/Team:Kent/Composite_Part"><li>Composite Parts</li></a>
+
                          
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<a href = "https://2017.igem.org/Team:Kent/Part_Collection"><li> Part Collection </li></a>
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                     </ul>
 
                     </ul>
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                     <ul class="drop-menu menu-2">
 
                     <ul class="drop-menu menu-2">
 
                         <a href="https://2017.igem.org/Team:Kent/Safety"><li>Project Safety</li></a>
 
                         <a href="https://2017.igem.org/Team:Kent/Safety"><li>Project Safety</li></a>
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                         <a href="https://2017.igem.org/Team:Kent/Signs"><li>Hazard Signs</li></a>
+
                          
 
                     </ul>
 
                     </ul>
 
                 </li>
 
                 </li>
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         </nav>
 
         </nav>
 
         <div id ="title">
 
         <div id ="title">
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                 <img src = "https://static.igem.org/mediawiki/2017/thumb/9/91/T--Kent--LogbookHeader.png/800px-T--Kent--LogbookHeader.png" id="header1">  
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                 <img src = "https://static.igem.org/mediawiki/2017/thumb/1/12/T--Kent--FutureHeader.png/800px-T--Kent--FutureHeader.png" id="header1">  
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<span>Logbook</span>
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<span>Future Ideas</span>
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<img src = "https://static.igem.org/mediawiki/2017/thumb/1/13/Logbookheader2.png/385px-Logbookheader2.png" id="header2">
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<img src = "https://static.igem.org/mediawiki/2017/thumb/7/7e/T--Kent--FutureHeader2v2.png/603px-T--Kent--FutureHeader2v2.png" id="header2">
 
         </div>
 
         </div>
 
         <nav class="droptext arrows">
 
         <nav class="droptext arrows">
 
<header class="hull">
 
<header class="hull">
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<label for="acc-close" class="hull-title">Basic Protocols</label>
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<label for="acc-close" class="hull-title">Due to the simple nature of our dCas13a protein construct, it can be utilized and ‘personalized’ for a
 +
variety of purposes. Our main objectives range from ‘near’ future, to ‘future’.</label>
 
</header>
 
</header>
 
<input type="radio" name="droptext" id="cb1" />
 
<input type="radio" name="droptext" id="cb1" />
 
<section class="hull">
 
<section class="hull">
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<label class="hull-title" for="cb1">Production of Lysogeny broth (LB)</label>
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<label class="hull-title" for="cb1">Near Future</label>
 
<label class="hull-close" for="acc-close"></label>
 
<label class="hull-close" for="acc-close"></label>
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<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
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<div class="hull-content">Although the processes within the central dogma have been studied and thoroughly characterized,
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litre of distilled water in a glass bottle. We then used a magnetic spinner to help mix the powders
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recent studies suggest mRNA localization is a way of controlling and regulating protein production. ¹ This
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with the water, we avoided shaking the glass bottle as it would cause froth and waste some of the
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mechanism appears to be similar to an induced suppressor that binds to DNA, thereby preventing its
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LB.
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transcription, except there are significantly more (regulating) factors involved. Unfortunately, our
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knowledge is very limited as mRNA’s dynamic nature makes it significantly more difficult to study.
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As our protein, LuCAS, has the ability to attach to, and track any RNA, given it has its complementary
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crRNA sequence, the main objective is to track and investigate mRNA localization. LuCAS is sensitive and
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selective to singular basepairs, making it a reliable tracking tool without altering the cell and its
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intracellular function itself.
 
<br>
 
<br>
 
When making the LB we also made another litre batch and added 15g of agar extract to be able to
 
When making the LB we also made another litre batch and added 15g of agar extract to be able to
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<input type="radio" name="droptext" id="cb2" />
 
<input type="radio" name="droptext" id="cb2" />
 
<section class="hull">
 
<section class="hull">
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<label class="hull-title" for="cb2">Production of SOB medium and magnesium stock</label>
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<label class="hull-title" for="cb2">Future</label>
 
<label class="hull-close" for="acc-close"></label>
 
<label class="hull-close" for="acc-close"></label>
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<div class="hull-content">Bringing together 20g of tryptone, 5g of yeast extract, 0.584g of NaCl, 0.186g of KCl and mixing it
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<div class="hull-content">In a variety of disease states, mRNA could be used as a ‘biomarker’ and therapeutic target. Oncogenes
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with 990ml of millipure water (using the magnetic mixer again) which was then put in to autoclave
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for example, such as Epidermal growth factor receptor (EGFR), are often overexpressed early in cancers
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to sterilise it, after it was taken out and let for it to cool down to below 60 o C.
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and treatment courses based on the type of mutations causing the cancer have been shown to be
−
<br>
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crucial to treatment success; in this case for example, tyrosine kinase inhibitors or monoclonal
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10ml of 2M Mg 2+ stock was then added and then brought to 100ml with millipure water, 0.2mm
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antibodies. Tools like LuCAS could potentially quantify and elucidate hyperproliferation and even qualify
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filter sterilize was then used</div>
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mRNA as therapeutic target to halt hyper proliferation of key ‘driver’ genes by targeting their mRNA.
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</section>
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Furthermore, monitoring mRNA expression in patients suffering chronic viral infections, such as HIV or
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<input type="radio" name="droptext" id="cb3" />
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hepatitis B, can help pinpoint disease progression and aid in treatment path selection, as disease
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<section class="hull">
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progression varies individually. HIV genes can be classified in early and late expression, with their
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<label class="hull-title" for="cb3">Production of SOC medium and glucose stock</label>
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detection of corresponding mRNA, via LuCAS, helping to narrow down the stage of disease.
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<label class="hull-close" for="acc-close"></label>
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The possible applications of LuCAS are many, as we simply propose a reliable and selective way to target
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<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
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mRNA in vivo without altering cellular functions; be it for research, diagnostics or therapeutics.
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bring 970 ml with millipure water and use the magnetic mixer once again, this was also then put in
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~ LuCas is still a toddler and these are his first baby steps, we can’t wait to see him prosper and grow
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to autoclave.
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into a young man that will change the world for the better</div>
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<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>
+
−
<input type="radio" name="droptext" id="acc-close" />
+
−
<input type="radio" name="droptext" id="cb4" />
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−
<section class="hull">
+
−
<label class="hull-title" for="cb4">Production of Glycerol stock</label>
+
−
<label class="hull-close" for="acc-close"></label>
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<div class="hull-content">If you wish to store bacteria long term, you will need to create a Glycerol Stock after
+
−
inoculating an overnight liquid culture
+
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<br>
+
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<ul><li>Once bacterial growth has been achieved, 500μL of the overnight liquid
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−
culture needs to be added to 500μL of 50% glycerol in a 2mL tube where it
+
−
should be gently mixed</li>
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−
<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>
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−
</li></ul></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="cb5" />
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<input type="radio" name="droptext" id="cb3" />
 
<section class="hull">
 
<section class="hull">
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<label class="hull-title" for="cb5">Running Agarose Gel</label>
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<label class="hull-title" for="cb3">References</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">Kejiou, N. S. and Palazzo, A. F. (2017), mRNA localization as a rheostat to regulate subcellular gene
−
<br>
+
expression. WIREs RNA, 8: n/a, e1416. doi:10.1002/wrna.1416</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>
+
−
<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
+
−
load into wells 2, 3, and 4 etc. (make sure you note what’s in each lane!)</li>
+
−
<li>Load all of your digests into the wells 2,3, and 4.</li>
+
−
<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>
+
−
<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>
 +
 
<input type="radio" name="droptext" id="acc-close" />
 
<input type="radio" name="droptext" id="acc-close" />
 
</nav>
 
</nav>

Latest revision as of 03:21, 16 December 2017


Future Ideas