Difference between revisions of "Team:Kent/Results"

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<label for="acc-close" class="hull-title">Basic Protocols</label>
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<div class="centerizer">
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</header>
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<span>iGEM Project Discription</span>
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<label class="hull-title" for="cb1">Production of Lysogeny broth (LB)</label>
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<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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litre of distilled water in a glass bottle. We then used a magnetic spinner to help mix the powders
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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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LB.
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<br>
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When making the LB we also made another litre batch and added 15g of agar extract to be able to
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grow bacteria on plates.</div>
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</section>
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<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-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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with 990ml of millipure water (using the magnetic mixer again) which was then put in to autoclave
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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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<br>
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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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filter sterilize was then used</div>
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</section>
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<section class="hull">
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<label class="hull-title" for="cb3">Production of SOC medium and glucose stock</label>
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<label class="hull-close" for="acc-close"></label>
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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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bring 970 ml with millipure water and use the magnetic mixer once again, this was also then put in
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to autoclave.
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<br>
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10ml of 2M Mg 2+ stock and then bring it to 100ml with milllipure water, filter sterilize it with 0.2m
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and then final add 20ml of 1M glucose stock.</div>
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</section>
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<section class="hull">
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<label class="hull-title" for="cb4">Production of Glycerol stock</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
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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
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should be gently mixed</li>
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<li>The glycerol stock should then be frozen at -80 o C<ul>
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<li> Successive freeze and thaw cycles will reduce the stocks shelf life</li></ul>
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</li></ul></div>
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</section>
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<label class="hull-title" for="cb5">Running Agarose Gel</label>
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<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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<br>
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<ul><li>Make up some agarose. This is done by taking 0.5g of agarose powder and putting it in a
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250ml sterile conical flask, with 50ml of TAE buffer, then microwaving it in small pulses (20
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seconds then swirling it around) until it is dissolved. Don’t overheat it or it will evaporate too
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much. Make up the evaporated volume to 50ml with distilled water.</li>
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<li>Add 1 vial of cybersafe (ask technical services for a tube of it and add all of it)</li>
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<li>Line the white sides of the tank with the agarose solution, to seal it and prevent leakage. Use
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a p1000 pipette set to 1ml. Let it dry (about 5 mins max)</li>
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<li>Then pour all the agarose/sybrsafe solution into the tank and put in the comb. Let it set and
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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
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tank with TAE buffer, so there’s at least half a centimetre of TAE covering the gel.</li>
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<li>Now, the samples need to be loaded. Load some DNA markers (ask technical services for a
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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
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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
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supply and drain the TAE buffer form the tank. Remove the gel with a spatula and place in a
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UV imaging box. Take an image of the gel under UV light, save it onto a USB stick.</li></ul></div>
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</section>
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<div id="textbox1" >
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<p>A multitude of genetically inherited and acquired diseases can be traced back to faulty or altered mRNA
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expression in eukaryotic cells. (1) To be able to treat these diseases and develop efficient therapeutic
 +
agents, the exact molecular mechanisms underlying faulty mRNA synthesis and export must be
 +
elucidated. Furthermore, studies of healthy cell proliferation and differentiation also indicate a degree
 +
of subcellular localization, which yet has to be investigated further to be explained and understood.(2)
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Our CRISPR/dCas13a construct functions as an “mRNA localization device” to investigate the fate of
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mRNA in vivo.
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</p>
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</div>
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<div class="centerizer">
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<span>dCAS13a</span>
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<div class="lineSeparator"></div>
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<div id="textbox2" >
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<p>Cas13a is a RNA guided endonuclease which degrades RNAs based on alignment of its CRISPR derived
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crRNA. We used dead Cas13a with four mutations, two in HEPN1 and two in the HEPN2 domains, which
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abort its nuclease activity, but maintain its helicase properties, resulting instead in attachment of
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dCAS13a to the targeted RNA sequence.
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</p>
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<img src="https://static.igem.org/mediawiki/2017/d/d0/T--Kent--construct.png">
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</div>
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<div class="centerizer">
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<span>gRNAs</span>
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<div class="lineSeparator"></div>
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<div id="textbox3" >
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<p>Simultaneously, guide RNAs constructs (for crRNA expression) destined for incorporation into the
 +
bacterial genome within CRISP repeats was designed. These constructs contain a U6 promoter followed
 +
by coding sequences for human β-Actin, pkp4, inpp-1, and Rab13 (individually) and terminated by an
 +
oligo (T) repeat. The protein sequences are chosen based on susceptibility to mutations and expression
 +
in disease states.
 +
</p>
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</div>
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<div class="centerizer">
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<span>Lucas</span>
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<div class="lineSeparator"></div>
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<div id="textbox4" >
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<p>With the aim to investigate and track mRNA localization in vivo, we engineered a gene construct
 +
containing a GFP attached via a linker sequence, allowing conformational flexibility, to a dCas13a coding
 +
gene. Additionally, two Nuclear localization sequences are on either side of the construct, which
 +
navigate the fusion protein back to the cell nucleus after protein synthesis in the ER is completed. In the
 +
nucleus, the protein construct recognizes the CRISPR incorporated sequence and attaches to it. When
 +
the targeted mRNA is consequently exported, the GFP+dCas13a tags along, allowing the ‘tracking’ of
 +
mRNA up until its translation in the ER. The unbound fusion protein constructs are retained in the
 +
nucleus and do not localize within the cell cytoplasm, reducing background noise.
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</p>
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</div>
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</div>
 
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Revision as of 03:53, 16 December 2017


Results
iGEM Project Discription

A multitude of genetically inherited and acquired diseases can be traced back to faulty or altered mRNA expression in eukaryotic cells. (1) To be able to treat these diseases and develop efficient therapeutic agents, the exact molecular mechanisms underlying faulty mRNA synthesis and export must be elucidated. Furthermore, studies of healthy cell proliferation and differentiation also indicate a degree of subcellular localization, which yet has to be investigated further to be explained and understood.(2) Our CRISPR/dCas13a construct functions as an “mRNA localization device” to investigate the fate of mRNA in vivo.

dCAS13a

Cas13a is a RNA guided endonuclease which degrades RNAs based on alignment of its CRISPR derived crRNA. We used dead Cas13a with four mutations, two in HEPN1 and two in the HEPN2 domains, which abort its nuclease activity, but maintain its helicase properties, resulting instead in attachment of dCAS13a to the targeted RNA sequence.

gRNAs

Simultaneously, guide RNAs constructs (for crRNA expression) destined for incorporation into the bacterial genome within CRISP repeats was designed. These constructs contain a U6 promoter followed by coding sequences for human β-Actin, pkp4, inpp-1, and Rab13 (individually) and terminated by an oligo (T) repeat. The protein sequences are chosen based on susceptibility to mutations and expression in disease states.

Lucas

With the aim to investigate and track mRNA localization in vivo, we engineered a gene construct containing a GFP attached via a linker sequence, allowing conformational flexibility, to a dCas13a coding gene. Additionally, two Nuclear localization sequences are on either side of the construct, which navigate the fusion protein back to the cell nucleus after protein synthesis in the ER is completed. In the nucleus, the protein construct recognizes the CRISPR incorporated sequence and attaches to it. When the targeted mRNA is consequently exported, the GFP+dCas13a tags along, allowing the ‘tracking’ of mRNA up until its translation in the ER. The unbound fusion protein constructs are retained in the nucleus and do not localize within the cell cytoplasm, reducing background noise.