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| − | .buttonblack {background-color: #3383C1; border-color: #3383C1; }
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| − | #protocolcontent {right: 20px; width: 63%;} .protcl {padding: 35 5 5 5;} #protocolcontent h2 {font-size: 16px; letter-spacing: 3px;}
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| − | @media(max-width:530px) { #menu2 {left: 10px; width: 28%;}}
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| − | @media(max-width:520px){ #menu3 {width: 80%; left: 10%; display: inline;} #menu2 {display: none;} #menu3 ul li {padding-left: 5px; padding-top: 3px; padding-bottom: 3px; padding-right: 10px; opacity: 0.8; font-size: 13px;}
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| − | #protocolcontent {width: 80%; right: 10%;} }
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| − | </style>
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| − | </head>
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| − | <body>
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| − | <div id="mobilebackground"></div>
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| − | <div id="header2">
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| − | <div id="wrapper">
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| − | <div id="menu2">
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| − | <p class="title" style="text-align:center;">Entrepreneurship</p>
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| − | <ul class="cos">
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| − | <li id="item1">Executive summary</li>
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| − | <li id="item2">Product</li>
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| − | <li id="item3">Intellectual Property</li>
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| − | <li id="item4">Manufacturing Operations</li>
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| − | <li id="item5">Business Environment</li>
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| − | <li id="item6">Marketing</li>
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| − | <li id="item7">Management</li>
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| − | <li id="item8">Financial Data</li>
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| − |
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| − | <!--
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| − | <li id="item9">IPTG-induced protein expression</li>
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| − | <li id="item10">Spectrophotometric assays</li>
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| − | <li id="item11">...</li>
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| − | <li id="item12">...</li>
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| − | <li id="item13">...</li> -->
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| − | </ul>
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| − | </div>
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| − | <div id="menu3">
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| − | <p class="title">Protocols</p>
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| − | <ul class="cos">
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| − | <a href="#summary"><li id="item1">Executive summary</li></a>
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| − | <a href="#product"><li id="item2">Product</li></a>
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| − | <a href="#IP"><li id="item3">Intellectual Property</li></a>
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| − | <a href="#Operations"><li id="item4">Manufacturing Operations</li></a>
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| − | <a href="#Marketing"><li id="item5">Business Environment</li></a>
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| − | <a href="#Management"><li id="item6">Marketing</li></a>
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| − | <a href="#Financialdata"><li id="item7">Management</li></a>
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| − | <a href="#Risk"><li id="item8">Financial Data</li></a>
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| − | <!--
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| − | <a href="#iptg"><li id="item9">IPTG-induced protein expression</li></a>
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| − | <a href="#assays"><li id="item10">Spectrophotometric assays</li></a>
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| − | <li id="item11">...</li>
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| − | <li id="item12">...</li>
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| − | <li id="item13">...</li> -->
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| − |
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| − | </ul>
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| − | </div>
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| − |
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| − | <div id="protocolcontent">
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| − | <div class="default-text"></div>
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| − | <div class="about-item1 hide">
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| − | <div class="protcl" id="summary"> <h2>Executive summary</h2>
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| − | <ol>
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| − | <li>Prepare restriction digestion mixture:
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| − | <ul><b>IDT gBlocks</b></ul>
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| − | <ul>- 10 ul of DNA (10 ng/ul)</ul>
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| − | <ul>- 2 ul of 10 x 2.1 buffer</ul>
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| − | <ul>- 0.3 ul of EcoR1</ul>
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| − | <ul>- 0.3 ul of Pst1</ul>
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| − | <ul>- 7.4 ul of milliQ H2O</ul><br/>
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| − |
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| − | <ul><b>Other digestions</b></ul>
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| − | <ul>- Required amount of DNA</ul>
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| − | <ul>- 1 ul of 10 x 2.1 buffer</ul>
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| − | <ul>- 0.3 ul of EcoR1</ul>
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| − | <ul>- 0.3 ul of Pst1</ul>
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| − | <ul>- add milliQ H2O up to 10 ul</ul>
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| − | <ul>(if using total volume greater than 10ul, increase the amount of buffer accordingly)</ul><br/>
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| − |
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| − | </li>
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| − | <li>Incubate at 37C for an hour</li>
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| − | <li> Heat inactivate by incubating at 80C for 20 minutes</li>
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| − | <li>Run a sample of digested DNA on a gel in order to confirm digestion:
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| − | <ul>- 2 ul of DNA</ul>
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| − | <ul>- 1 ul of 6 x Gel Loading Dye</ul>
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| − | <ul>- 3 ul of milliQ H2O</ul>
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| − | </li>
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| − | <li>If digestion is confirmed, proceed to ligation </li>
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| − | </ol>
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| − | </div>
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| − |
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| − | </div>
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| − |
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| − |
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| − | <div class="about-item2 hide">
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| − |
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| − | <div id="product" class="protcl"> <h2>Product</h2>
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| − | <p> </p>
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| − | <h3> Overview </h3>
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| − | <p>ProKao is a delicious chocolate bar that combines the goodness of chocolate and the benefits of probiotics.
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| − | Our chocolate is sourced from organic and fair trade producers, and has been enhanced with billions of
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| − | friendly bacteria, which have been shown to interact with your gut-brain axis, to make you happier and healthier,
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| − | and help you cope with the stress of everyday life.</p>
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| − | <p>Once a day you will be able to indulge in our delicious chocolate bar, knowing that not only it will deliver
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| − | these healthy bacteria to your gut, but also contains dietary fibers (prebiotics), which stimulate the
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| − | proliferation of your natural microflora, without any added calories.</p>
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| − |
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| − | <img src="https://static.igem.org/mediawiki/2015/2/23/UCL2015product3infographic.jpeg"> </li>
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| − |
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| − | <h3>How our product came to be</h3>
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| − | <p>The idea to create a probiotic to help with stress, anxiety and other mental health-related issues came
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| − | from the core theme of our project: the gut-brain axis. Our original idea was to create genetically engineered
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| − | probiotic strains but soon we realised that the current legislation doesn’t allow for the commercialisation of
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| − | such products aimed at the general public, and only recently have tests with animals began taking place for
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| − | pharmaceutical applications.</p>
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| − |
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| − | <h3>A three tier vision</h3>
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| − | <p>The set of constraints we were initially faced with lead us to pivot away from focusing genetically engineered
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| − | probiotics from the start. Instead we opted for the following timeline:</p>
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| − | <ol>
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| − | <li>A product based on characterised strains and other GRAS (generally recognised as safe) ingredients that have
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| − | a track record of efficacy and clinical data to back them up. This will be our minimum viable product,
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| − | categorised as a food.</li>
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| − | <li>Once the mechanisms behind the effect of different microbiome profiles have been fully identified it will be
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| − | possible to create enhanced strains. This can be done without necessarily being subjected to the tight
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| − | regulations that restrict genetically engineered products, of course this would limit the scope of our action
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| − | to certain techniques (deregulation, generating mutations, directed evolution, CRISPR-cas9). Another option
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| − | would be to run our own experiments on particular strains we create or discover and patent them. These products
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| − | would also be characterised as foods.</li>
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| − | <li>Using synthetic biology methods to enhance the production of particular compounds, introduce the genetic
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| − | circuits that we have created to respond to environmental triggers and create ways of controlling the production of particular substances.
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| − | This phase offers the greatest potential and the widest variety of applications; bacteria can be engineered
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| − | into microbial factories that produce pharmacologically active substances of our choosing and secrete them in
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| − | the desired amounts. A product such as this one is beyond the scope of what current legislation allows, and
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| − | would only be considered for approval as a therapeutic and therefore be subjected to clinical
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| − | trials.</li>
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| − | </ol>
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| − |
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| − | <img src="https://static.igem.org/mediawiki/2015/4/4d/Ucl2015entrepprodthree.jpeg">
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| − |
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| − | <h3> Why Chocolate? </h3>
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| − | <p>Independent studies have shown chocolate to be 3 to 4 times more effective in delivering probiotics to your
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| − | gut than dairy products, as it protects them from being degraded in the passage through the stomach.</p>
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| − | <p>It is a delicious treat that will not only benefit your gut flora, but also has all these added benefits:</p>
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| − | <ul>
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| − | <li> <h4> Reduces the risk of cardiovascular disease </h4> </li>
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| − | <p>There are hundreds of studies that provide supporting evidence that chocolate improves the functioning of the
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| − | cardiovascular system, due to its high content in antioxidants. </p>
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| − | <p> The flavanols in dark chocolate have been shown to increase the production of Nitric Oxide (NO), which makes
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| − | the arteries relax, lowering blood pressure, and facilitating blood flow (Fraga, 2011). This also improves
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| − | athletic performance, and recovery due to its anti-inflammatory properties.</p>
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| − | <p>In addition to this, chocolate promotes the creation of good cholesterol, which in the long term reduces
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| − | the cholesterol accumulation in the arteries. (Buijsse, 2006)</p>
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| − | <li> <h4>Improve Brain function and enhance mood </h4> </li>
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| − | <p> Dark chocolate contains compounds such as anandamide, PEA, theobromine and tryptophan, that increase serotonin
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| − | and endorphin levels, which, in combination with our added probiotics, boost overall mood, and make you feel
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| − | happier by encouraging the release of certain neurotransmitters such as melatonin. The consequences are increased
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| − | libido, and an antidepressant effect.</p>
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| − | <p>The high flavonols content also improves memory and cognitive function, allowing you to be more aware, keep
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| − | a sharp mind and a positive attitude (Scholey, 2013) </p>
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| − | <li> <h4> It contains a high concentration of minerals </h4>
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| − | <p>100g of Quality Dark Chocolate (70-85%) contains:</p>
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| − | <ul> - 67% of the RDA for Iron.</ul>
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| − | <ul> - 58% of the RDA for Magnesium. </ul>
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| − | <ul> - 89% of the RDA for Copper. </ul>
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| − | <ul> - 98% of the RDA for Manganese. </ul>
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| − | <ul> - plenty of potassium, phosphorus, zinc and selenium.</ul>
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| − | <p>These are essential in building muscles, optimizing energy production, and are necessary for your
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| − | immune function.</p>
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| − | </li>
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| − | </ul>
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| − |
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| − | <h3> References </h3>
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| − | <ul>
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| − | <li>Buijsse, Brian, Edith JM Feskens, Frans J Kok, and Daan Kromhout. 2006. Cocoa intake, blood pressure, and
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| − | cardiovascular mortality: the Zutphen Elderly Study. Archives of internal medicine 166, no. 4: 411-417.</li>
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| − | <li>Fraga, César G, María C Litterio, Paula D Prince, et al. 2011. Cocoa flavanols: effects on vascular nitric
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| − | oxide and blood pressure. Journal of clinical biochemistry and nutrition 48, no. 1: 63</li>
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| − | <li>Scholey, Andrew, and Lauren Owen. 2013. Effects of chocolate on cognitive function and mood: a systematic
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| − | review.Nutrition reviews 71, no. 10: 665-681.</li>
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| − | </ul>
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| − |
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| − | </div>
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| − | </div>
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| − |
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| − |
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| − | <div class="about-item3 hide">
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| − | <div id="IP" class="protcl"><h2>Intellectual property</h2>
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| − | <h4>
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| − | <ol>
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| − | <li>(If using part from the distribution: resuspend the DNA in 10 ul of MiliQ water, making sure that it turns red. Wait 10 minutes before adding the DNA to cells)</li>
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| − | <li>Put a tube of NEB DH 5 alpha <i>E. coli</i> cells on ice and wait until they thaw completely. Divide the cells into 50 ul aliquotes. </li>
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| − | <li>Add 1 ul of plasmid DNA to 50 ul of cells. </li>
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| − | <li>Mix by carefully flicking the tube. Do not vortex or pipette in and out! </li>
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| − | <li>Place the mixture on ice for 30 minutes. </li>
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| − | <li>Heat shock the cells at 42 °C for 30 seconds and immediately put on back on ice.</li>
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| − | <li>Keep cells on ice for next 5 minutes. Do not mix. </li>
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| − | <li>Pipette 950 ul of SOC media kept at room temperature into the mixture. If SOC is not available, use LB. <br/>
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| − | <li>Incubate the mixture at 37 °C for 60 minutes </li>
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| − | <li>Prepare plates with appropriate antibiotics. Bring plates to room temperature before plating. Use 2 plates per transformation reaction. </li>
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| − | <li>Plate 200 ul of cells on one plate.
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| − | <li>Pellet the remaining cells and resuspend in 200ul of LB.</li>
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| − | <li> Plate the remaining cells on second plate.</li>
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| − | <li>Incubate plates overnight at 37 °C. </li>
| |
| − | </ol>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − |
| |
| − | <div class="about-item4 hide">
| |
| − |
| |
| − | <div id="operations" class="protcl"><h2>Manufacturing Operations</h2>
| |
| − | <div id="submenu" style="padding-top: 30px;">
| |
| − | <a data-scroll data-options='{ "easing": "linear" }' href="#1" class="buttonblack">Prototype</a>
| |
| − | <a data-scroll data-options='{ "easing": "linear" }' href="#2" class="buttonblack">Production</a>
| |
| − | <a data-scroll data-options='{ "easing": "linear" }' href="#3" class="buttonblack">Stratified</a>
| |
| − | <a data-scroll data-options='{ "easing": "linear" }' href="#4" class="buttonblack">Personalised</a>
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| − | <a data-scroll data-options='{ "easing": "linear" }' href="#5" class="buttonblack">References</a>
| |
| − | <!-- <a data-scroll data-options='{ "easing": "linear" }' href="#4" class="buttonblack">Developers</a -->
| |
| − | </div>
| |
| − |
| |
| − | <h3 id="1" style="padding-top:50px;">Prototype</h3>
| |
| − | <p>With our product implementation plan in place, and a solid business model, we have developed a prototype
| |
| − | with our engineered bacteria to test how effective the chocolate matrix is in encapsulating the bacteria and ensuring
| |
| − | their survival for an effective delivery.</p>
| |
| − |
| |
| − | <h4>Protocol</h4>
| |
| − | <ol>
| |
| − | <li>Innoculate in two different Falcon tubes Nissle- TPH1 20ml of LB + Cam and shake overnight at 250rpm and
| |
| − | 37ºC</li>
| |
| − | <li>Centrifuge at 40000rpm for 7min and discard the media.</li>
| |
| − | <li>Resuspend the pellet of one of the tubes in a solution composed of 1g of sugar and 1ml of miliQ water (A),
| |
| − | and the one of the other in 500ul of miliQ water (B)</li>
| |
| − | <li> Melt 50g of dark chocolate (52%). The product also contained: Cocoa butter, Whey powder, Soya Lecithin,
| |
| − | Polyglycerol polyricinoleate, and vanilla extract.</li>
| |
| − | <li>Mix 10g of chocolate with 500ul of the solution A (labeled 1.50 in the photo), and solution B (labeled
| |
| − | 2.50). </li>
| |
| − | <li>Mix 10g of chocolate with 250ul of the solution A (labeled 1.25), and solution B (labeled 2.25)</li>
| |
| − | <li>Mix 10g of chocolate with 50ul of mili Q water for the Control</li>
| |
| − | <p>Note: the chocolate must be at under 40ºC before mixing with bacterial solution</p>
| |
| − | <li>Leave samples in the fridge overnight</li>
| |
| − | <figure>
| |
| − | <img src="https://static.igem.org/mediawiki/2015/0/0a/Ucl2015entrepprototypesolid.jpg" style="width:564px;height:345px">
| |
| − | </figure>
| |
| − | <li>Cut 5g of sample and wrap in aluminum foil for storage, to test shelf life</li>
| |
| − | <figure>
| |
| − | <img src= "https://static.igem.org/mediawiki/2015/5/5c/Ucl2015entrepprototypeshelfs.jpg" style="width:500px;height:381px">
| |
| − | </figure>
| |
| − | <li>Cut 2g of sample and add to 5ml of LB in a falcon tube</li>
| |
| − | <li>Water bathe falcon tubes at 39ºC until the chocolate melts and mix by vortexing until a homogeneous
| |
| − | texture is achieved</li>
| |
| − | <li>plate 100ul of solution in Cam LB agar plates and incubate overnight at 37ºC</li>
| |
| − | </ol>
| |
| − | <p> The bacteria grew back on the agar plates after having been placed on the chocolate matrix overnight, which
| |
| − | showed chocolate is an adequate media to place them. In addition, we observed a higher number of CFU in the plate
| |
| − | of solution B, suggesting the more effective method for resuspension of the pellet. </p>
| |
| − | <figure>
| |
| − | <img src= "https://static.igem.org/mediawiki/2015/4/4e/Ucl2015entrepprototypepetri.jpg" style="width:600px;height:373px">
| |
| − | </figure>
| |
| − |
| |
| − | <p> However, from literature we know that the delivery and shelf life is best if the bacteria are microencapsulated
| |
| − | beforehand with methods such as spray drying. There is also literature showing that when tested in a simulator of
| |
| − | the human digestive system, probiotics in a chocolate matrix proved to reach the gut more effectively than those
| |
| − | in Dairy (Possemiers, 2010)</p>
| |
| − | <p> This is the first step towards a more sophisticated prototype, in which we will test freeze dried bacteria
| |
| − | against other types of microencapsulation, to optimize the delivery, as we want to be able to guarantee that at
| |
| − | least 2 billion CFUs reach the gut and colonize it. </p>
| |
| − | <p> Our finalized chocolate bar will be a 50g single dose rectangular bar. </p>
| |
| − | <figure>
| |
| − | <img src= "https://static.igem.org/mediawiki/2015/f/f6/Ucl2015entrepprototypewrap.jpg" style="width:400px;height:444px">
| |
| − | </figure>
| |
| − |
| |
| − | <h3 id="2">Scale-up Production</h3>
| |
| − | <p>Manufacturing will be facilitated by outsourcing the production of our ingredients to key partners with
| |
| − | experience in the sector. These partners will provide the already processed chocolate, and encapsulated
| |
| − | probiotics. They will be selected according to the quality criteria with which the product identifies.
| |
| − | This is mainly organic fair trade cocoa pods, and other more specific certifications to cater to the needs of
| |
| − | specific sectors of the general public, such as a gluten free certification or dairy free.</p>
| |
| − | <p>This will be beneficial to us, as these companies can off-set early production, which will allow our products
| |
| − | to reach the market sooner. This allows us to focus on the further research of the biological components needed
| |
| − | for the development of the company to the second and third stages.</p>
| |
| − | <p> For the first stages of the business development, we would use strains whose effect is documented in literature</p>
| |
| − | <figure>
| |
| − | <img src= " https://static.igem.org/mediawiki/2015/d/d7/Ucl2015entreptablestrains.jpg " style="width:500px;height:446px">
| |
| − | </figure>
| |
| − |
| |
| − | <p>Production will take place following two different models, for two slightly different products</p>
| |
| − | <h4 id="3"> Stratified </h4>
| |
| − | <p>In the stratified model, consumers identify themselves with a specific category, and select the chocolate bar
| |
| − | that is more adequate to suit their needs. </p>
| |
| − | <p> Whereas most probiotics companies we found offered a range of different products based on flavours we would
| |
| − | target market segments instead. We will produce a fixed number of bars with specific probiotic contents,
| |
| − | specifically designed for each market segment. These will be differentiated in terms of age, sex, and
| |
| − | lifestyle; which will lead to strata like: higher protein content for more athletic people, or a limited
| |
| − | amount of lactobacillus strains for vegans.</p>
| |
| − | <p>This model has a simpler production, given that it’s a repetitive process in which machines can be programmed
| |
| − | for a certain amount of hours to produce one specific type of bar, and then switch to a different one by just
| |
| − | changing the proportions to be added, and producing a great amount.</p>
| |
| − |
| |
| − | <h4 id="4"> Personalised </h4>
| |
| − | <p>The personalized model, adds a layer of complexity to the structure. It will consist of made to order
| |
| − | individual batches of probiotic chocolate tailored to the users goals and needs to in a way that has never
| |
| − | been done before. </p>
| |
| − | <figure>
| |
| − | <img src="https://static.igem.org/mediawiki/2015/f/f9/Ucl2015entrepmicroprofile.jpeg" width="304" height="228">
| |
| − | <figcaption>Simulation of customer's microbiome profile</figcaption>
| |
| − | </figure>
| |
| − |
| |
| − | <p> Using information supplied to us by the user we could design a probiotic fit specifically for their profile
| |
| − | and monitor its effect by monitoring the state of the microbiome periodically. Over time more metrics and
| |
| − | applications would be added, optimizing our ability to cater for each specific profile, and ultimately
| |
| − | fulfilling the promise of power over all the potential that microscopic factories living within us can offer.</p>
| |
| − |
| |
| − | <h4 id="5"> References </h4>
| |
| − | <ul>
| |
| − | <li>Bravo JA, Forsythe P, Chew MV et al. (2011) Ingestion of Lactobacillus strain regulates emotional behavior
| |
| − | and central GABA receptor expression in a mouse via the vagus nerve. Proc Natl Acad Sci USA 108, 16050–16055.</li>
| |
| − | <li>Dapoigny M, Piche T, Ducrotte, Lunaud B, Cardot JM, Bernalier-Donadille A (2012): Efficacy and safety
| |
| − | profile of LCR35 complete freeze –dried culture in irritable bowel syndrome: A randomized , double blind
| |
| − | study. World J Gastroenterol 18:2067-2075.</li>
| |
| − | <li>Desbonnet L, Garrett L, Clarke G et al. (2008) The probiotic Bifidobacteria infantis: an assessment of
| |
| − | potential antidepressant properties in the rat. J Psychiatr Res 43, 164–174.</li>
| |
| − | <li>Hsiao EY, McBride SW, Hsien S et al. (2013) Microbiota behavioral and physiological abnormalities
| |
| − | associated with neurodevelopmental disorders. Cell 155, 1451–1463.</li>
| |
| − | <li>Ko CY, Lin HTV & Tsai GJ (2013) Gamma-aminobutyric acid production in black soybean milk by Lactobacillus
| |
| − | brevis FPA 3709 and the antidepressant effect of the fermented product on a forced swimming rat model.
| |
| − | Process Biochem 48, 559–568. </li>
| |
| − | <li>Messaoudi M, Lalonde R, Violle N, Javelot H, Desor D, Nejidi A, et al. (2011): Assessment of
| |
| − | psychotropic-like properties of a probiotic formulation (Lactobacillus helveticus R0052 and
| |
| − | Bifidobacterium longum R0175) in rats and human subjects Br J Nutr 105:755-764 </li>
| |
| − | <li>Possemiers, Sam et al. (2010). Bacteria and chocolate: a successful combination for probiotic
| |
| − | delivery."International journal of food microbiology 141.1: 97-103. </li>
| |
| − | <li>Rao AV, Bested AC, Beaulne TM, Katzman MA, Iorio C, Berardi JM, Logan AC (2009): A randomized,
| |
| − | double-blind, placebo-controlled pilot study of a probiotic in emotional symptoms of chronic fatigue
| |
| − | syndrome. Gut Pathog 1:6. </li>
| |
| − | </ul>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="about-item5 hide">
| |
| − | <div id="environment" class="protcl"><h2>Business Environment</h2>
| |
| − | <div id="submenu" style="padding-top: 30px;">
| |
| − | <a data-scroll data-options='{ "easing": "linear" }' href="#1" class="buttonblack">SWOT Analysis</a>
| |
| − | <a data-scroll data-options='{ "easing": "linear" }' href="#2" class="buttonblack">PESTLE Analysis</a>
| |
| − | <a data-scroll data-options='{ "easing": "linear" }' href="#3" class="buttonblack">Regulations</a>
| |
| − | <!-- <a data-scroll data-options='{ "easing": "linear" }' href="#4" class="buttonblack">Developers</a -->
| |
| − | </div>
| |
| − |
| |
| − | <p id="1">text</p>
| |
| − | <h4>
| |
| − | <ol>
| |
| − | <li>Digest at least 500 ng of each part according to the restriction digestion</li>
| |
| − | <li>Run the digested DNA on the gel according to gel electrophoresis protocol</li>
| |
| − | <li>Identify the parts that you want to ligate on a gel and cut the bands out using razor blade</li>
| |
| − | <li>Purify the excised bands using the commercial kit according to the manufacturer's instructions</li>
| |
| − | <li>Quantify the DNA yield using DNA nanodrop</li>
| |
| − | <li>Proceed to ligation</li>
| |
| − |
| |
| − | </ol>
| |
| − | </h4>
| |
| − | </div></div>
| |
| − |
| |
| − | <div class="about-item6 hide">
| |
| − | <div id="marketing" class="protcl"><h2>Marketing</h2>
| |
| − | <div id="submenu" style="padding-top: 30px;">
| |
| − | <a data-scroll data-options='{ "easing": "linear" }' href="#1" class="buttonblack">Stratified - B2B</a>
| |
| − | <a data-scroll data-options='{ "easing": "linear" }' href="#2" class="buttonblack">Personalised - B2C</a>
| |
| − | <!-- <a data-scroll data-options='{ "easing": "linear" }' href="#3" class="buttonblack">Developers</a -->
| |
| − | </div>
| |
| − |
| |
| − | <p id="1">text</p>
| |
| − | <ol>
| |
| − | <li>Prepare the PCR mix:
| |
| − | <ul>- 12.5 ul of 2 x Q5 PCR master mix</ul>
| |
| − | <ul>- 1.25 ul of 10 uM forward primer</ul>
| |
| − | <ul>- 1.25 ul of 10 uM reverse primer</ul>
| |
| − | <ul>- 2 ng of DNA to be PCRed</ul>
| |
| − | <ul>- add milliQ H2O up to 25 </ul>
| |
| − | </li>
| |
| − | <li> Set up the PCR cycles according to the following rules:
| |
| − | <ul><b>Initial denaturation</b></ul>
| |
| − | <ul>- 98C for 30 seconds</ul>
| |
| − |
| |
| − | <ul><b>35 cycles</b></ul>
| |
| − | <ul>- 98C for 10 seconds</ul>
| |
| − | <ul>- 30 seconds at primer melting temperature</ul>
| |
| − | <ul>- 72C for 30sec/kb of PCRed fragment</ul>
| |
| − |
| |
| − | <ul><b>Final extension</b></ul>
| |
| − | <ul>- 72C for 2 minutes</ul>
| |
| − | <ul>- Hold at 4C</ul>
| |
| − | </li>
| |
| − | <li>Confirm the PCR by running 2 ul of the product on the gel according to the gel electrophoresis protocol</li>
| |
| − | </ol>
| |
| − | </div></div>
| |
| − |
| |
| − |
| |
| − |
| |
| − | <div class="about-item7 hide">
| |
| − | <div id="management" class="protcl"><h2>Management</h2>
| |
| − |
| |
| − | <h4>
| |
| − | <p>When it comes to small companies and startups, the organizational structure has to be designed to be the most
| |
| − | efficient way for the company to achieve its goals and objectives. </p>
| |
| − | <p>Since our company has a long term plan of several parallel projects, we decided for a matrix structure where all
| |
| − | can be worked on in a parallel way, without getting the streams of communication to interfere with each other. </p>
| |
| − | <img src="https://static.igem.org/mediawiki/2015/c/c1/UCL2015entrepmanagement.jpeg">
| |
| − | <h3>Functional Management</h3>
| |
| − | <p>We decided to give our management a functional structure, centered in job functions and departments, in blue.
| |
| − | These will be overseen by the C’s suite. They will all report to the board of directors thus centralizing the
| |
| − | decision making, which is essential in startups.</p>
| |
| − | <p>Functional structures are specifically designed to work in heavily project- focused companies, like ours.
| |
| − | Directors will assign each of the projects to a manager, who will coordinate the whole development, getting
| |
| − | support from each department in each aspect of the project development. This allows each project manager to be
| |
| − | specialized in their stream and effectively meet deadlines, thus ensuring a more rapid business development,
| |
| − | essential in the ever-changing conditions of the current market.</p>
| |
| − |
| |
| − | <h3>Matrix Organisation</h3>
| |
| − | <p>We specifically selected the matrix model because we consider that it’s the one that best allows the horizontal
| |
| − | flow of skills and information, which, is essential in such a multidisciplinary endeavor. </p>
| |
| − | <p>It is common in the development phases, as it allows for large projects to move forward in a more
| |
| − | organized way, since it allows employees to specialize in a project and apply their expertise in a particular
| |
| − | field, without being removed from their department in question.</p>
| |
| − | <p>The project manager’s authority flows horizontally across departmental boundaries, but is always subjected
| |
| − | to the board of directors.</p>
| |
| − |
| |
| − | <h3>Leadership</h3>
| |
| − | <p>In terms of leadership we have advocated for a Collaborative Leadership model, which encourages equal
| |
| − | participation across all levels, therefore giving the project managers the most responsibility on the decision
| |
| − | making and problem solving, almost at a CEO level.</p>
| |
| − | <p>This would imply open information sharing across the company, as every department as to be on the same
| |
| − | page about each project, thus ensuring optimum efficiency in interdepartmental collaborations. This cross
| |
| − | departmental participation allows more creative approaches to problem solving, and idea generation, speeding
| |
| − | up once more business development.</p>
| |
| − | <p> Resource allocation will be managed by the board of directors. Team leaders will request these proactively
| |
| − | and provide them to their teams, for which a fluent communication is key. This ensures a fast project advance
| |
| − | as the employees have all resources available to do their jobs efficiently.</p>
| |
| − | <p>This type of structure places more responsibility on each team member, which makes them more involved in the
| |
| − | process, and means issues are dealt with swiftly. The fact that leaders and team members are valued equally
| |
| − | creates a better working environment and a better flow of feedback, which allows the team to be in a constant
| |
| − | state of improvement; and employees to constantly acquire new knowledge and experience. This is essential to
| |
| − | respond to the challenging and highly competitive environment that current market poses. </p>
| |
| − | <p>This structure responds to employees’ seek for autonomy and engagement, and promotes professional
| |
| − | satisfaction, and a better environment for innovation. </p>
| |
| − |
| |
| − |
| |
| − |
| |
| − | </ol>
| |
| − | </div></div>
| |
| − |
| |
| − | <div class="about-item8 hide">
| |
| − | <div id="financialdata" class="protcl"><h2>Financial Data</h2>
| |
| − | <div id="submenu" style="padding-top: 30px;">
| |
| − | <a data-scroll data-options='{ "easing": "linear" }' href="#1" class="buttonblack">Prototype</a>
| |
| − | <a data-scroll data-options='{ "easing": "linear" }' href="#2" class="buttonblack">Production</a>
| |
| − | <!-- <a data-scroll data-options='{ "easing": "linear" }' href="#3" class="buttonblack">Developers</a -->
| |
| − | </div>
| |
| − |
| |
| − | <p id="1">text</p>
| |
| − | <ol>
| |
| − |
| |
| − | </ol>
| |
| − |
| |
| − | </div></div>
| |
| − |
| |
| − | <!--<div class="about-item9 hide">
| |
| − | <div id="iptg" class="protcl"><h2>IPTG-induced protein expression</h2>
| |
| − | <h4>
| |
| − | <ol>
| |
| − |
| |
| − | <li>On the afternoon before the induction, start seed culture with appropriate antibiotic from glycerol stock and leave to incubate overnight at 37C shaking</li>
| |
| − | <li>The next morning, use 2 µl of seed culture to inoculate 100 ml of fresh media with appropriate antibiotic in shaker flask and grow until an OD600 nm of 0.4-0.6 is reached</li>
| |
| − | <li>If necessary, prepare these in the meantime :
| |
| − | <ul>- 50 ml stock of lysis buffer (25 mM TRIS-Cl, 2 mM EDTA, pH 7.6):
| |
| − | <li> weight 0.151g of Tris base</li>
| |
| − | <li>add 45 ml of water</li>
| |
| − | <li>titrate with HCL to pH 7.6</li>
| |
| − | <li>fill up to 50 ml with water</li>
| |
| − | <li>add 29.2 mg of EDTA</li></ul>
| |
| − | <ul>- IPTG 100 mM stocks: (23.8 mg IPTG per 1ml ddH2O )</ul>
| |
| − | <li>When culture reaches OD of 0.4-0.6, add IPTG to a final concentration of 1 mM (=100ul of 100 mM stock)</li>
| |
| − | <li>Incubate induced culture at 30 °C for 4 hours</li>
| |
| − | <li>Split the culture into 4 falcon tubes (~25 ml each) and harvest the pellets by centrifugation for 20mins at max speed</li>
| |
| − | <li>Resuspend each pellet in 2 ml of lysis buffer, lyse by sonication (10 cycles of 10 sec with 10 sec breaks)</li>
| |
| − | <li>Centrifuge 20 min at max speed.</li>
| |
| − | <li>Transfer all supernatants to separate tube</li>
| |
| − | <li>Measure the concentration of protein in supernatant using Bradford Assay</li>
| |
| − | <li>Store in the freezer</li>
| |
| − |
| |
| − |
| |
| − |
| |
| − |
| |
| − | </ol></h4>
| |
| − | </div></div>
| |
| − |
| |
| − |
| |
| − | <div class="about-item10 hide">
| |
| − | <div id="assays" class="protcl"><h2>Spectrophotometric assays</h2>
| |
| − | <h4>
| |
| − | <ol>
| |
| − | - Assay for tryptophan hydroxylase (<b>BBa_K1598002</b>) activity: <a href="https://static.igem.org/mediawiki/2015/c/cb/UCL_BBa_K1598002_Assay.pdf">download protocol</a>
| |
| − |
| |
| − | </ol>
| |
| − | </h4>
| |
| − | </div></div>
| |
| − | -->
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| − |
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| − |
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| − | </div></div></div>
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