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time consuming, and expertise requiring method. This led us to develop our own measurement method, OraCell. {% endblock %}
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<h2>Intro</h2>
<h1>Intro</h1>
<hr>
<p>Our project is centered around the production of the secondary metabolite decursin. The system includes the three
penultimate reactions in the original synthesis pathway of decursin. This involves two enzymatic reactions and
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As a side note, in Hebrew the word “Or” means light and since we’re utilizing changes in luminescence this made
the choice of name even more appropriate. </p>
<h2>The Hippo Pathway</h2>
<h1>The Hippo Pathway</h1>
<hr>
<p> While researching decursin, we discovered that in addition to being the leading candidate in treatment for chemotherapy-induced
alopecia, it significantly assists in the inhibition of tumor growth<sup>[<ahref="#ref3">3</a>]</sup>. One possible mechanism for its tumor
suppression capabilities is through the <b>Hippo signaling pathway</b><sup>[<ahref="#ref4">4</a>]</sup>. In this pathway, Yap/Taz proteins translocate
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elements
</p>
</div>
<h2>OraCell</h2>
<h1>OraCell</h1>
<hr>
<p>The OraCell system is based on an existing plasmid with the Luciferase reporter gene, downstream of TEAD response
elements (Addgene plasmid <ahref="http://n2t.net/addgene:34615">#34615</a>). Transfection of this plasmid
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<br>
<h2>Experiments & Methods</h2>
<h1>Experiments & Methods</h1>
<hr>
<p>The main pillars of the workflow used to establish the “OraCell” stable cell-line are composed of multiple experiments
and protocols devised based on knowledge provided by our mentors and our literature review.</p>
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</p>
</div>
<h3>Plasmid Construction</h3>
<h2>Plasmid Construction</h2>
<p>We used the 8xGTIIC-Luciferase plasmid as a backbone for our plasmid system (Addgene plasmid <ahref="http://n2t.net/addgene:34615">#34615</a>). It includes a pGL3b vector and the genes encoding for Luciferase downstream of GTIIC motifs
with high affinity for TEAD transcription factors. This was convenient due to the abundance of prior results.
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<h3>Preliminary calibration experiments </h3>
<h2>Preliminary calibration experiments </h2>
<p>Once we verified that our plasmid was ready to be transfected to the mammalian cells, we were able to continue
to the next stage in the workflow.</p><p> We chose to work with Chinese hamster ovary (CHO) cells, because they are easily
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acquainted with the protocols of splitting, seeding and transfection, and find optimal parameters such as the
time it takes plates to reach confluence and the optimum concentration of the transfection reagents . </p>
<h5>Seeding calibration</h5>
<h4>Seeding calibration</h4>
<p>CHO cells were grown in 10cm plates, but experiments were performed in 6-well plates. Due to time constraints,
an optimal seeding density of a 6-well plate was sought, such that overnight incubation would result in a confluent
plate on the next day.</p><p> 6-well plates were seeded with a different cell density ranging from 10<sup>4</sup> cells /cm<sup>2</sup> to
10<sup>5</sup> cells/cm<sup>2</sup>. After overnight incubation we counted the number of cells in each plate using a counting chamber.
<p>Our chosen method of transfection was PEI. This transfection reagent is used to introduce the plasmid DNA to the
host cell, it condenses DNA into positively charged particles that are endocytosed and transferred to the cytoplasm.
<sup>[<ahref="#ref8">8</a>]</sup> PEI was chosen due to its availability and low cost. Stock solution concentration was 1mg/ml.</p><p>
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would express BFP that in turn expresses fluorescence.</p><p></p> 2.5 μg of BFP plasmid was mixed with increasing amounts
of PEI and used FACS to measure the cell’s viability and fluorescence 48 hours post-transfection. </p>
<h5>Stable Integration</h5>
<h2>Stable Integration</h5>
<p>To establish a stable clonal cell line, cells were transfected with the blasticidin resistance gene (BlastR)-modified
Luciferase plasmid and subjected to a high concentration of blasticidin. Then, we used FACS (Bigfoot Spectral
Cell Sorter) to perform single-sorting cell into individual wells on a plate and allowed cells to proliferate
before testing their Luciferase expression with luciferin.</p>
<h3>Luciferase Assay</h3>
<h2>Luciferase Assay</h2>
<p>To measure the transfected cell reporter gene, Luciferase, we performed a luminescence assay. This was done both
to validate the transfection itself, and to study how decursin affects the gene expression.</p><p> We used a Biotium’s
Firefly Luciferase Assay Kit 2.0, cells were lysed, and luciferin was added to the lysate. This resulted in an
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that contains the genes encoding for both Luciferase and luciferin. </p>
<h1>Results</h1>
<hr>
<p> Our initial cloning, calibrations and luminescence experiments can be found on our <ahref="{{ url_for('pages', page='results') }}">results</a> page.
Following these preliminary results, we began adding decursin and measuring the luminescence to investigate
if there was indeed a decrease. The first of these experiments was a time calibration experiment to see under
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decursin and if decursin is truly able to interfere with the Hippo pathway, regardless of the mode.</p>
<h1> Prospects </h1>
<hr>
<p>The OraCell assay was shown to be a efficient, simple and high-throughput method that holds the potential of detecting
and quantifying a variety of molecules. The method still needs a few more experiments to verify its capabilities,
which should hopefully be accomplished in the coming weeks to show its potential utility for groups all across
