Difference between revisions of "Team:DTU-Denmark/products"

 
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                         <h1>Title<p class="lead">leader under the title, short introduction. Ubique moderatius efficiantur eum et, dico oporteat recusabo ius cu, pro id modus sadipscing. Maluisset patrioque eum ad, mel eius doctus accommodare eu, minimum deleniti repudiandae mel ea. Noster nostrud diceret sea no. Eos an nullam molestiae signiferumque, vel ne laudem ignota oblique. Duo te luptatum percipitur signiferumque, at dicunt iriure dolorem his.</p></h1>
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                         <h1>Products<p class="lead">In the Molecular tools section we developed several molecular tools, potentially allowing us to genetically engineer <i><i>Yarrowia lipolytica</i></i>. In this section we apply these tools and attempt to use these to achieve expression in <i>Y. lipoltyca</i>.</p></h1>
 
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                             <p>“Productivity is never an accident. It is always the result of a commitment to excellence, intelligent planning, and focus efforts”
                             <small>Someone famous in <cite title="Source Title">Source Title</cite></small>
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                             <small>Paul J. Meyer, <cite title="Source Title">It's your life. Live big.</cite></small>
 
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                 <p>“Productivity is never an accident. It is always the result of a commitment to excellence, intelligent planning, and focus efforts”<sup><a href="#references"> 3</a></sup></p>
                 <small>Someone famous in <cite title="Source Title">Source Title</cite></small>
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                 <small>Paul J. Meyer, <cite title="Source Title">It's your life. Live big.</cite><</small>
 
             </blockquote>
 
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             <p>
 
             <p>
By developing tools to genetically engineer Yarrowia lipolytica we aim to create a versatile cell factory, which in the future will be able to produce almost any desired product ranging from complex therapeutic proteins to high value chemical compounds. Besides diversity of products cell factory based on Yarrowia lipolytica offers a wide range of substrate tolerance.  
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                By developing tools to genetically engineer <i>Yarrowia lipolytica</i> we aim to create a versatile cell factory, which in the future will be able to produce almost any desired product ranging from complex therapeutic proteins to high value chemical compounds. Besides diversity of products cell factory based on <i>Y. lipolytica</i> offers a wide range of substrate tolerance.  
</p><p>
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            </p>
We aim to demonstrate this versatility of our Y. lipolytica strain as a cell factory by producing an extracellular heterologous protein such as proinsulin. Moreover to prove compliance of our cell factory with the iGEM BioBricks standards we aim to produce beta-carotene from the registered BioBricks. In order to introduce genes necessary to achieve both of mentioned ideas we apply pSB1A8yl plasmid, constructed by our team.  
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            <p>
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                We aim to demonstrate this versatility of our <i>Y. lipolytica</i> strain as a cell factory by producing heterologous proteins. Our first attempt is the production of humanized, <i>Renilla reniformis</i> GFP (hrGFP), followed by proinsulin. To achieve both of mentioned ideas we apply the pSB1A8YL plasmid, cloning workflow and codon optimization algorithm developed by our team.  
 
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         <h2 class="h2">Proinsulin</h2>
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         <h2 class="h2">hrGFP</h2>
            <h3 class="h3">Role of insulin in our bodies</h3>
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                <p>
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Insulin is a peptide hormone that plays a crucial role in glucose homeostasis and prevents harmful levels of sugar in the blood. After a meal, beta-cells of pancreas islets release insulin to the blood stream. Then insulin activates the glucose transporters, present on the cells surface, and cells are able to absorb the glucose.
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            <h3 class="h3">Insulin biosynthesis</h3>
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                <p>
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                    At the first stage insulin is synthesized as a chain of 101 amino acids, called preproinsulin, which comprises of signal peptide (pre-peptide) and three short chains: A, B and C. The pre-peptide, responsible for directing a nascent polypeptide, is removed from preproinsulin giving proinsulin. Subsequently, the proinsulin is folded and two disulphide bonds are created between chain A and B and one links chain A. In the last step, the C chain is digested from the proinsulin by an exoprotease - carboxypeptidase E [5]. The mature insulin contains chains A and B linked by 3 disulphide bonds and in total comprises of 51 amino acids.
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                </p><p>
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                    Insulin is linked with diabetes mellitus, the disease that affects insulin production and results in too high sugar level in the blood stream. The treatment is based on taking insulin from the external sources.
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                </p>
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            <h3 class="h3">Goal</h3>
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                <p>
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                    In order to demonstrate Yarrowia lipolytica as a versatile cell factory we aim to produce proinsulin as an answer to increasing global problem with diabetes.
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                </p>
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            <h3 class="h3">Numbers of diabetes</h3>
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                <p>
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Current estimations point out that nowadays 8.5% of the global population stuffers from diabetes (which corresponds to 422 million people). Even more disturbing are the predictions, that show that the diabetes will affect 14% of global population by 2040 will (corresponds to 642 million people, and assuming global population to rise to 9.16 billion) [3].
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                </p><p>
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                    In 2014 the global insulin market reached $24 billion and it will double by 2020 achieving level of $48 billion.
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                </p><p>
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Denmark has a long tradition of producing insulin to treat diabetes. Novo Nordisk, the company established in Denmark in 1923, is one of the main insulin produces. Nowadays insulin production is based on recombinant technology, which means that insulin gene is introduced to the host organism, either prokaryotic (Escherichia coli) or eukaryotic (Saccharomyces cerevisiae). This approach brings both opportunities as possibility of modifying the coding signals, as limits, since post-translation modifications cannot be carried out in prokaryotic host and production in S. cerevisiae gives lover yield than E.coli. 
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</p>
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             <h3 class="h3">Design</h3>
 
             <h3 class="h3">Design</h3>
 
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                 <p>
The human proinsulin sequence was obtain from Sures et al. (1980)). The sequence was codon optimized for Y. lip using the codon optimization tool developed by our team.  
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            In order to assess if we were able to follow our cloning flow proposed in the molecular tools section which would be expressed in <i>Y. lipolytica</i>, we chose to develop our own BioBricks. Namely a constitutive transcription factor alpha (TEF1) promoter native to <i>Y. lipolytica</i> (<a href="http://parts.igem.org/Part:BBa_K2117000">BBa_K2117000</a>) and the hrGFP gene (<a href="http://parts.igem.org/Part:BBa_K2117003">BBa_K2117003</a>) previously used successfully in <i>Y. lipolytica</i><sup><a href="#references">1</a></sup>. The hrGFP sequence was codon optimized by the algorithm developed by our team. By combining these two parts in a device (<a href="http://parts.igem.org/Part:BBa_K2117005">BBa_K2117005</a>) the expression should in theory be easily detected due to the fluorescence signal produced. The cloning flow can be seen in Figure 1.
In order to ensure the high rate of transcription we applied the native promoter of Y. lipolytica, TEF1.  
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            </p>
By adding the iGEM standard RFC10 prefix and suffix to both proinsulin gene and pTEF we were able to clone them into the standard backbones as well as in our Y. lipolytica shuttle vector using either 3A assembly, Gibson assembly or USER cloning.
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The literature presents several methods of insulin detection that also seem valid for proinsulin. Screening for the recombinant protein may be performed using SDS-PAGE for the cell lysate. Western blotting is another alternative, widely used for insulin detection.
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            <img id="img1" class="enlarge img-responsive figure-img" src="https://static.igem.org/mediawiki/2016/thumb/f/f8/T--DTU-Denmark--Cloning_flow2.png/800px-T--DTU-Denmark--Cloning_flow2.png" alt="DESCRIPTION">
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            <br>
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            <figcaption class="figure-caption"><strong>Figure 1:</strong> Cloning flow of the expression test of pSB1A8YL in <i>Y. lipolytica</i>. The expression of the GFP should yield a flourescent ouput detectable by fluorescence microscopy or fluorometer measurements.</figcaption>
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             <h3 class="h3">Results</h3>
 
             <h3 class="h3">Results</h3>
                <p>
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            <p>
                    We succeed to create a construct comprised of the shuttle vector pSB1A8yl with the TEF promoter and the codon optimized proinsulin. The construct was transformed into Y. lip. The slowered growth was observed thus the expression of heterologous protein may be suspected. Component detection was performed by the SDS-PAGE and Western blotting. however none of these methods gave a satisfying results. We suspect that SDS-PAGE failed due to the low detection level. As mentioned before Western blotting is suitable for insulin, however proinsulin is not exported through ER thus it is missing in a secretion signal and as a result may not be folded properly. As a solution another approach could be used, mainly fusion of the proinsulin with either GFP or His followed by a detection of an attached fluorescent protein or peptide instead of the proinsulin itself.
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            The parts were ordered as gBlocks and assembled in <i>E. coli</i> using A3 assembly. The assembly was confirmed using restriction analysis (See Figure 2), PCR and sequencing (data not shown).
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            Afterwards the construct was transformed into <i>Y. lipolytica</i> PO1f and grown on plates containing selective media. Single colonies were picked and grown in liquid selective media, and subjected to fluorescence microscopy. Figure 3 shows the <i>Y. lipolytica</i> PO1f cells under a confocal laser microscope with 100x magnification. The high fluorescent output from with the <a href="http://parts.igem.org/Part:BBa_K2117005">BBa_K2117005</a> construct, proves that the cells are producing hrGFP, and ultimately that our expression system consisting of pSB1A8YL and the TEF1 promoter (<a href="http://parts.igem.org/Part:BBa_K2117000">BBa_K2117000</a>) can be used for heterologous protein expression in <i>Y. lipolytica</i>.
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            <img id="img4" class="enlarge img-responsive figure-img" src="https://static.igem.org/mediawiki/2016/thumb/9/99/T--DTU-Denmark--TEF%2BGFP_restriction_analysis.jpg/246px-T--DTU-Denmark--TEF%2BGFP_restriction_analysis.jpg" alt="DESCRIPTION" width="157">
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            <br>
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            <figcaption class="figure-caption"><strong>Figure 2:</strong> Analytical digestion of the pSB1A8YL containing the <a href="http://parts.igem.org/Part:BBa_K2117005">BBa_K2117005</a> device. The fragment lengths can be seen on the ladder, and the restriction enzyme and predicted fragment lengths is stated above the fragments.</figcaption>
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            <img id="img2" class="enlarge img-responsive figure-img" src="https://static.igem.org/mediawiki/2016/6/60/T--DTU-Denmark--GFP_Tef%2BhrGFP_all.png" alt="DESCRIPTION">
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            <img id="img3" class="enlarge img-responsive figure-img" src="https://static.igem.org/mediawiki/2016/4/40/T--DTU-Denmark--GFP_control.png" alt="DESCRIPTION" width="478">
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            <br>
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            <figcaption class="figure-caption"><strong>Figure 3:</strong> Fluorescence microscopy conducted by a confocal laser microscope with 100x magnification. A and D are taken using standard brightfield, B and E are taken using the GFP filter and with the excitation laser on and C and F are overlays of the two photos where the black bagground has been removed (C is an overlay of A and B, and F is an overlay of D and E). A, B and C are <i>Y. lipolytica</i> PO1f cells with our GFP expressing device (<a href="http://parts.igem.org/Part:BBa_K2117005">BBa_K2117005</a>) shuttled by our plasmid pSB1A8YL. D, E and F are <i>Y. lipolytica</i> PO1f cells with the empty pSB1A8YL plasmid, which serves as a control for the GFP signal. Notice that even though the empty vector control shows trace amounts of auto-fluoresence the strain with the GFP expressing device clearly exhibits higher levels of fluorescence, which proves that our expression system works as intended.</figcaption>
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        <p style="color:#FFFFFF;">.</p>
 
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            As seen in Figure 3 our construct produced a strong GFP signal, which ultimately proves that our expression system works as intended.
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         <div><a class="anchor" id="section-3"></a>
 
         <div><a class="anchor" id="section-3"></a>
         <h2 class="h2">Beta-carotene</h2>
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         <h2 class="h2">Insulin</h2>
             <p>
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             Our experiments told us that our plasmid was a powerful tool, allowing us to conveniently assembling constructs in <i>E. coli</i>, which would be expressed in <i>Y. lipolytica</i>. Now we wanted to use it to take on the ultimate challange: Could we use this tool to produce proinsulin, and potentially increase the sustainability of insulin production?
Vitamin A has been proved to be important for eyesight in mammals, but we do not have the full pathway for the synthesis of this essential vitamin. Beta-carotene is an important precursor, which must be obtained through the diet.
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             <h3 class="h3">Theory</h3>
            </p>
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             <h3 class="h3">Numbers of vitamin A</h3>
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                 <p>
 
                 <p>
Deficiency of vitamin A affects in the majority of cases children and pregnant women. WHO reports that every year 190 million children suffer from deficiency of this vitamin and 5,2 million among these children struggle with night blindness. This numbers may explain the demand for vitamin A. Estimations for this year demonstrates that the carotenoid market will reach $1.24 billion. It is expected that the carotenoid market will achieve $1.8 billion by 3 years (2019) [7].  
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                    Insulin is a peptrode hormone synthesised in the pacreas, and plays a crucial role in glucose homeostasis and prevents harmful levels of sugar in the blood. Beta-cells in the islets of the pancreas release insulin to the blood stream, which stimulates muscle fat and liver cells to absorb and store glucose from the bloodstream by interacting with insulin-specific receptors<sup><a href="#references">2</a></sup>. Diabetes is caused by either immune-mediated destruction of pacreatic islet cells (type 1 diabetes), or pancreatic exhaustion causing insulin resistance (type 2 diabetes). Both types of diabetes is cured by artificially controlling the insulin concentration in the patient by injection<sup><a href="#references">3</a></sup>. The treatment of diabetes with insulin was discovered in 1921, and its ability to restore health is so dramatic, that it was initially described in terms such as "the rising of the dead". Considering this it perhaps does not come as a surprise that insulin is often described as one of the most important biomedical events of the last century<sup><a href="#references">4</a></sup>, and as the number of adults with diabetes is expected to rise from 415 million today to 642 million in 2040, the relevance of diabetes is higher than ever<sup><a href="#references">6</a></sup>. To read more about the insulin market, see our <a href = "https://2016.igem.org/Team:DTU-Denmark/description">descriptions page</a>.
Besides increasing demand for vitamin A there is also another motivation, mainly way of the production. Vitamin A has so far mostly been produced by artificial chemical synthesis or by extraction from carrots, however the benefits of using microorganisms for production on an industrial scale is increasing (Barredo 2012).
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                 </p>
 
                 </p>
             <h3 class="h3">Biosynthesis of the beta-carotene</h3>
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             <h3 class="h3">Insulin biosynthesis</h3>
 
                 <p>
 
                 <p>
Beta-carotene is naturally produced by a range of organisms such as plants and fungi, but neither conventional yeast nor Y. lipolytica has the pathway for biosynthesis. Beta-carotene is produced by four enzymatic steps from farnesyl diphosphate (F-PP), which is naturally produced in Y. lipolytica. In the next step, farnesyl diphosphate is converted to geranylgeranyl diphosphate (GG-PP) in a reaction catalyzed by geranylgeranyl diphosphate synthase (CrtE). GG-PP is transformed to phytoene by CrtYB, which is an enzyme with two domains, one functioning as phytoene synthase and another as lycopene cyclase, in this reaction the first domain plays a crucial role. The next step results in production of lycopene and is catalyzed by carotene desaturase (CrtI). Finally, lycopene is converted by CrtYB with the lycopene cyclase domain into beta-carotene [6].
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                    At the first stage insulin is synthesized as a chain of 101 amino acids, called preproinsulin with a molecular weight of 12 Kilo daltons (kDa), which comprises of signal peptide (pre-peptide) and three short chains: A, B and C. The pre-peptide, responsible for directing a nascent polypeptide, is removed from preproinsulin giving the proinsulin consisting of 86 amino acids with a molecular weight of 9.4kDa. Subsequently, the proinsulin is folded and two disulphide bonds are created between chain A and B and one links chain A. In the last step, the C chain is digested from the proinsulin by an exoprotease - carboxypeptidase E. The mature insulin contains chains A and B linked by 3 disulphide bonds and in total comprises of 51 amino acids with a molecular weight of 5.8kDa<sup><a href="#references">5</a></sup>. The maturation of preproinsulin into proinsulin and finally mature insulin is shown in Figure 4.
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                    <br>
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                    <figcaption class="figure-caption"><strong>Figure 4:</strong> Schematic representation of the maturation of preproinsulin into proinsulin and finally mature insulin. The colors represents the following: Pink: Signal peptide, yellow: B chain, green: C chain, Blue: A chain, red: Disulphide bonds. The proteolytic events is carried out by the carboxypeptidase E. Figure from <a href=" http://watcut.uwaterloo.ca/webnotes/Metabolism/Hormones.html#hormonesInsulinwatcut.com">WatCut</a>.</figcaption>
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                    The first insulin was extracted from animals, and <i>E. coli</i> was emplyoed in the production of the A and B chain separately as recombinant DNA technologies progessed. Today insulin is produced in <i>Saccharomyces cerevisiae</i> by a single chain nonnative "miniproinsulin" precursor, which is matured by trypsin and carboxypeptidase B<sup><a href="#references">4</a></sup>.
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                <p> <b>
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                    Goal</b><br>
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                    In order to demonstrate <i>Y. lipolytica</i> as a versatile cell factory we aim to produce proinsulin as an answer to increasing global demand for insulin.
 
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             <h3 class="h3">Design</h3>
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             <h3 class="h3">Results</h3>
 
                 <p>
 
                 <p>
The JHU 2011 iGEM team successfully produced beta-carotene in Saccharomyces cerevisiae by constructing three biobricks with the three individual genes encoding the enzymes from the pathway from the fungi Xanthophyllomyces dendrorhous.
+
                    The human proinsulin sequence was obtained from Sures et al. (1980). The sequence was codon optimized for the codon bias of <i>Y. lipolytica</i> using the codon optimization tool developed by our team, and ordered as a gBlock. The proinsulin gene with the RFC10 prefix and suffix was amplified by PCR into our BioBrick <a href="http://parts.igem.org/Part:BBa_K2117003">BBa_K2117003</a> and assembled with our TEF1 promoter to form our device <a href="http://parts.igem.org/Part:BBa_K2117002">BBa_K2117002</a>. This was done in pSB1A8YL using standard A3 assembly. The construct was transformed into <i>E. coli</i>, purified and confirmed using analytical digenstion, PCR and sequencing (data not shown). After confirmation of the construct, it was transformed into <i>Y. lipolytica</i>. The transformants was subjected to colony PCR (see Figure 5). The cloning flow can be seen in Figure 6.
</p><p>
+
In order to remove illegal restriction sites from the beta-carotene biobricks we perform site directed mutagenesis. By designing primers for Gibson assembly of the CrtE, CrtI and CrtYB we combine all three genes with the Y. lip ribosome binding site (RBS), CACA, in front of each ORF. The created construct can be transformed to Y. lip using the shuttle plasmid pSB1A8yl. Since cells overproducing beta-carotene change their color to orange no sophisticated detection method is required.  
+
 
                 </p>
 
                 </p>
            <h3 class="h3">Results</h3>
+
                  <center>
 +
                    <figure class="figure">
 +
                        <img id="img5" class="enlarge img-responsive figure-img" src="https://static.igem.org/mediawiki/2016/thumb/e/e0/T--DTU-Denmark--TEF%2BProins_restriction_analysis_png.png/800px-T--DTU-Denmark--TEF%2BProins_restriction_analysis_png.png" alt="DESCRIPTION" width="450">
 +
                        <br>
 +
                        <figcaption class="figure-caption"><strong>Figure 5:</strong> Analytical digestion of the pSB1A8YL containing the <a href="http://parts.igem.org/Part:BBa_K2117002">BBa_K2117002</a> device. The fragment lengths can be seen on the ladder, and the primers predicted fragment lengths is stated above the fragments.</figcaption>
 +
                    </figure>
 +
                    </center>
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                    <!-- The Modal with same picture-->
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                        <img class="modal-content" id="img5Img">
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                        <div class="caption">DESCRIPTION</div>
 +
                    </div>
 +
                    <center>
 +
                    <figure class="figure">
 +
                        <img id="img6" class="enlarge img-responsive figure-img" src="https://static.igem.org/mediawiki/2016/thumb/d/de/T--DTU-Denmark--Cloning_flow3.png/800px-T--DTU-Denmark--Cloning_flow3.png" alt="DESCRIPTION">
 +
                        <br>
 +
                        <figcaption class="figure-caption"><strong>Figure 6:</strong> The cloning flow of the proinsulin producing strain.</figcaption>
 +
                    </figure>
 +
                    </center>
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                        <img class="modal-content" id="img6Img">
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                        <div class="caption">DESCRIPTION</div>
 +
                    </div>
 
                 <p>
 
                 <p>
We improved currently existing biobricks for beta-carotene genes: CrtE, CrtI and CrtYB by inserting ribosome binding site – CACA, in from of each of the gene and removing illegal restriction sites.  
+
                    We knew that detecting the proinsulin would be the biggest challange yet, as all previous detections was based on chromoproteins and fluorescence, while this was a protein without any intrinsic detectable signal. We had several considerations for detecting the proinsulin production including rtPCR and ELISA, altough ended up chosing SDS page and Western blotting, as these methods provide excellent sensitivity while some of the experiences running the SDS page experiments could be transfered to the western blotting experiments. The results of these experiments are show in Figure 7 and 8.
 +
                </p> 
 +
                <center>
 +
                <figure class="figure">
 +
                        <img id="img7" class="enlarge img-responsive figure-img" src="https://static.igem.org/mediawiki/2016/thumb/9/9b/T--DTU-Denmark--Proins_SDS.png/800px-T--DTU-Denmark--Proins_SDS.png" alt="DESCRIPTION" width="600">
 +
                        <br>
 +
                        <figcaption class="figure-caption"><strong>Figure 7:</strong> SDS page of <i>Y. lipoltyica</i> cell lysate stained with Coomassie blue. The construct the cells were transformed with and the dilution factor of the crude protein extract can be seen above the bands. The weight of the proteins can be seen on the ladders. Notice that no distinct band is visible at the approximate size of proinsulin (9.4kDa).</figcaption>
 +
                    </figure>
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                        <img class="modal-content" id="img7Img">
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                        <div class="caption">DESCRIPTION</div>
 +
                    </div>
 +
                <center>
 +
                <figure class="figure">
 +
                        <img id="img8" class="enlarge img-responsive figure-img" src="https://static.igem.org/mediawiki/2016/thumb/f/f5/T--DTU-Denmark--Proins_WB.jpg/716px-T--DTU-Denmark--Proins_WB.jpg" alt="DESCRIPTION" width="500">
 +
                        <br>
 +
                        <figcaption class="figure-caption"><strong>Figure 8:</strong> Western Blotting of <i>Y. lipoltyica</i> cell lysate. The lysates were loaded the same way as the SDS page gel above. Notice that only noise is present on the picture.</figcaption>
 +
                    </figure>
 +
                    </center>
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                    <!-- The Modal with same picture-->
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                        <img class="modal-content" id="img8Img">
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                        <div class="caption">DESCRIPTION</div>
 +
                    </div>
 +
 
 +
                    <p>
 +
                        Unfortunately, non of these results gave conclusive evidence for the presence of proinsulin in our samples. The lack of proinsulin on the SDS could be caused by the concentration of proinsulin being lower than the detection limit. The lack of signal in the western blotting results could be caused by <a href="http://www.abcam.com/insulin-antibody-epr3075-ab108326-references.html">our antibody</a> not recognising the proinsulin produced due to incomplete folding, or sub-optimal treatment of the membrane. Of course there is also the possibility that our strain is not producing proinsulin, although due to the positive results using hrGFP we are confident that our expression system works. Other effects such as mRNA folding or rapid protein degradation may also be factors leading to us not being able to detect proinsulin in the samples. Future efforts could include a fusion of the proinsulin, a reporter (such as GFP) or a purification tag (such as a His-tag) aiding the detection of proinsulin.
 +
                    </p>
 +
        </div>
 +
       
 +
       
 +
       
 +
        <div><a class="anchor" id="section-4"></a>
 +
            <h2 class="h2">Conclusion</h2>
 +
                <p>
 +
                    In this section we apply the tools made in the molecular toolbox section, for heterologous protein production in <i>Y. lipolytica</i>. This is done by combining two of our own BioBricks, a constitutive transcription factor alpha (TEF1) promoter native to <i>Y. lipolytica</i> (<a href="http://parts.igem.org/Part:BBa_K2117000">BBa_K2117000</a>) and the hrGFP gene (<a href="http://parts.igem.org/Part:BBa_K2117003">BBa_K2117003</a>) codon optimized by the algorithm developed by our team. By combining these two parts using A3 assembly and the cloning flow suggested in the molecular tools section, we created the device <a href="http://parts.igem.org/Part:BBa_K2117005">BBa_K2117005</a>. By confirming the production of hrGFP, we were able to prove that heterologous protein production is possible when our tool pSB1A8YL. Even though efforts to translate this into proinsulin production failed, the modular nature of pSB1A8YL means that it has potential to become a powerful tool when using <i>Y. lipolytica</i> for research or indsutrial purposes.
 
                 </p>
 
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 +
        <!-- Reference section -->
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 +
            <h2 class="h2">References</h2>
 +
            <ol>
 +
                <li>Blazeck, J., Liu, L., Redden, H., & Alper, H. (2011). Tuning Gene Expression in Yarrowia lipolytica by a Hybrid Promoter Approach. Applied and Environmental Microbiology, 77(22), 7905–7914. doi:10.1128/AEM.05763-11</li>
 +
                <li><a href="https://www.niddk.nih.gov/health-information/diabetes/types/prediabetes-insulin-resistance">NIDDK</a></li>
 +
                <li>Zaykov, A. N., Mayer, J. P., & DiMarchi, R. D. (2016). Pursuit of a perfect insulin. Nat Rev Drug Discov, 15(6), 425–439. JOUR. Retrieved from http://dx.doi.org/10.1038/nrd.2015.36</li>
 +
                <li>Mayer, J. P., Zhang, F., & DiMarchi, R. D. (2007). Insulin structure and function. Biopolymers, 88(5), 687–713. doi:10.1002/bip.20734</li>
 +
                <li>Baeshen, N. A., Baeshen, M. N., Sheikh, A., Bora, R. S., Ahmed, M. M. M., Ramadan, H. A. I., … Redwan, E. M. (2014). Cell factories for insulin production. Microbial Cell Factories, 13(1), 141. doi:10.1186/s12934-014-0141-0</li>
 +
                <li><a href="http://www.idf.org/sites/default/files/IDF_AnnualReport_2015_WEB.pdf">IDF annual report 2015</a></li>
 +
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             <li><a href="#section-1">Section 1</a></li>
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             <li><a href="#section-1">Introduction</a></li>          
             <li><a href="#section-2">Section 2</a></li>
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             <li><a href="#section-2">hrGFP</a></li>
             <li><a href="#section-3">Section 3</a></li>
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             <li><a href="#section-3">Insulin</a></li>
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Latest revision as of 01:38, 20 October 2016

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Products

In the Molecular tools section we developed several molecular tools, potentially allowing us to genetically engineer Yarrowia lipolytica. In this section we apply these tools and attempt to use these to achieve expression in Y. lipoltyca.


Introduction

“Productivity is never an accident. It is always the result of a commitment to excellence, intelligent planning, and focus efforts” 3

Paul J. Meyer, It's your life. Live big.<

By developing tools to genetically engineer Yarrowia lipolytica we aim to create a versatile cell factory, which in the future will be able to produce almost any desired product ranging from complex therapeutic proteins to high value chemical compounds. Besides diversity of products cell factory based on Y. lipolytica offers a wide range of substrate tolerance.

We aim to demonstrate this versatility of our Y. lipolytica strain as a cell factory by producing heterologous proteins. Our first attempt is the production of humanized, Renilla reniformis GFP (hrGFP), followed by proinsulin. To achieve both of mentioned ideas we apply the pSB1A8YL plasmid, cloning workflow and codon optimization algorithm developed by our team.

hrGFP

Design

In order to assess if we were able to follow our cloning flow proposed in the molecular tools section which would be expressed in Y. lipolytica, we chose to develop our own BioBricks. Namely a constitutive transcription factor alpha (TEF1) promoter native to Y. lipolytica (BBa_K2117000) and the hrGFP gene (BBa_K2117003) previously used successfully in Y. lipolytica1. The hrGFP sequence was codon optimized by the algorithm developed by our team. By combining these two parts in a device (BBa_K2117005) the expression should in theory be easily detected due to the fluorescence signal produced. The cloning flow can be seen in Figure 1.

DESCRIPTION
Figure 1: Cloning flow of the expression test of pSB1A8YL in Y. lipolytica. The expression of the GFP should yield a flourescent ouput detectable by fluorescence microscopy or fluorometer measurements.

Results

The parts were ordered as gBlocks and assembled in E. coli using A3 assembly. The assembly was confirmed using restriction analysis (See Figure 2), PCR and sequencing (data not shown).

Afterwards the construct was transformed into Y. lipolytica PO1f and grown on plates containing selective media. Single colonies were picked and grown in liquid selective media, and subjected to fluorescence microscopy. Figure 3 shows the Y. lipolytica PO1f cells under a confocal laser microscope with 100x magnification. The high fluorescent output from with the BBa_K2117005 construct, proves that the cells are producing hrGFP, and ultimately that our expression system consisting of pSB1A8YL and the TEF1 promoter (BBa_K2117000) can be used for heterologous protein expression in Y. lipolytica.

DESCRIPTION
Figure 2: Analytical digestion of the pSB1A8YL containing the BBa_K2117005 device. The fragment lengths can be seen on the ladder, and the restriction enzyme and predicted fragment lengths is stated above the fragments.
DESCRIPTION
DESCRIPTION
Figure 3: Fluorescence microscopy conducted by a confocal laser microscope with 100x magnification. A and D are taken using standard brightfield, B and E are taken using the GFP filter and with the excitation laser on and C and F are overlays of the two photos where the black bagground has been removed (C is an overlay of A and B, and F is an overlay of D and E). A, B and C are Y. lipolytica PO1f cells with our GFP expressing device (BBa_K2117005) shuttled by our plasmid pSB1A8YL. D, E and F are Y. lipolytica PO1f cells with the empty pSB1A8YL plasmid, which serves as a control for the GFP signal. Notice that even though the empty vector control shows trace amounts of auto-fluoresence the strain with the GFP expressing device clearly exhibits higher levels of fluorescence, which proves that our expression system works as intended.

.

As seen in Figure 3 our construct produced a strong GFP signal, which ultimately proves that our expression system works as intended.

Insulin

Our experiments told us that our plasmid was a powerful tool, allowing us to conveniently assembling constructs in E. coli, which would be expressed in Y. lipolytica. Now we wanted to use it to take on the ultimate challange: Could we use this tool to produce proinsulin, and potentially increase the sustainability of insulin production?

Theory

Insulin is a peptrode hormone synthesised in the pacreas, and plays a crucial role in glucose homeostasis and prevents harmful levels of sugar in the blood. Beta-cells in the islets of the pancreas release insulin to the blood stream, which stimulates muscle fat and liver cells to absorb and store glucose from the bloodstream by interacting with insulin-specific receptors2. Diabetes is caused by either immune-mediated destruction of pacreatic islet cells (type 1 diabetes), or pancreatic exhaustion causing insulin resistance (type 2 diabetes). Both types of diabetes is cured by artificially controlling the insulin concentration in the patient by injection3. The treatment of diabetes with insulin was discovered in 1921, and its ability to restore health is so dramatic, that it was initially described in terms such as "the rising of the dead". Considering this it perhaps does not come as a surprise that insulin is often described as one of the most important biomedical events of the last century4, and as the number of adults with diabetes is expected to rise from 415 million today to 642 million in 2040, the relevance of diabetes is higher than ever6. To read more about the insulin market, see our descriptions page.

Insulin biosynthesis

At the first stage insulin is synthesized as a chain of 101 amino acids, called preproinsulin with a molecular weight of 12 Kilo daltons (kDa), which comprises of signal peptide (pre-peptide) and three short chains: A, B and C. The pre-peptide, responsible for directing a nascent polypeptide, is removed from preproinsulin giving the proinsulin consisting of 86 amino acids with a molecular weight of 9.4kDa. Subsequently, the proinsulin is folded and two disulphide bonds are created between chain A and B and one links chain A. In the last step, the C chain is digested from the proinsulin by an exoprotease - carboxypeptidase E. The mature insulin contains chains A and B linked by 3 disulphide bonds and in total comprises of 51 amino acids with a molecular weight of 5.8kDa5. The maturation of preproinsulin into proinsulin and finally mature insulin is shown in Figure 4.

DESCRIPTION
Figure 4: Schematic representation of the maturation of preproinsulin into proinsulin and finally mature insulin. The colors represents the following: Pink: Signal peptide, yellow: B chain, green: C chain, Blue: A chain, red: Disulphide bonds. The proteolytic events is carried out by the carboxypeptidase E. Figure from WatCut.

The first insulin was extracted from animals, and E. coli was emplyoed in the production of the A and B chain separately as recombinant DNA technologies progessed. Today insulin is produced in Saccharomyces cerevisiae by a single chain nonnative "miniproinsulin" precursor, which is matured by trypsin and carboxypeptidase B4.

Goal
In order to demonstrate Y. lipolytica as a versatile cell factory we aim to produce proinsulin as an answer to increasing global demand for insulin.

Results

The human proinsulin sequence was obtained from Sures et al. (1980). The sequence was codon optimized for the codon bias of Y. lipolytica using the codon optimization tool developed by our team, and ordered as a gBlock. The proinsulin gene with the RFC10 prefix and suffix was amplified by PCR into our BioBrick BBa_K2117003 and assembled with our TEF1 promoter to form our device BBa_K2117002. This was done in pSB1A8YL using standard A3 assembly. The construct was transformed into E. coli, purified and confirmed using analytical digenstion, PCR and sequencing (data not shown). After confirmation of the construct, it was transformed into Y. lipolytica. The transformants was subjected to colony PCR (see Figure 5). The cloning flow can be seen in Figure 6.

DESCRIPTION
Figure 5: Analytical digestion of the pSB1A8YL containing the BBa_K2117002 device. The fragment lengths can be seen on the ladder, and the primers predicted fragment lengths is stated above the fragments.
DESCRIPTION
Figure 6: The cloning flow of the proinsulin producing strain.

We knew that detecting the proinsulin would be the biggest challange yet, as all previous detections was based on chromoproteins and fluorescence, while this was a protein without any intrinsic detectable signal. We had several considerations for detecting the proinsulin production including rtPCR and ELISA, altough ended up chosing SDS page and Western blotting, as these methods provide excellent sensitivity while some of the experiences running the SDS page experiments could be transfered to the western blotting experiments. The results of these experiments are show in Figure 7 and 8.

DESCRIPTION
Figure 7: SDS page of Y. lipoltyica cell lysate stained with Coomassie blue. The construct the cells were transformed with and the dilution factor of the crude protein extract can be seen above the bands. The weight of the proteins can be seen on the ladders. Notice that no distinct band is visible at the approximate size of proinsulin (9.4kDa).
DESCRIPTION
Figure 8: Western Blotting of Y. lipoltyica cell lysate. The lysates were loaded the same way as the SDS page gel above. Notice that only noise is present on the picture.

Unfortunately, non of these results gave conclusive evidence for the presence of proinsulin in our samples. The lack of proinsulin on the SDS could be caused by the concentration of proinsulin being lower than the detection limit. The lack of signal in the western blotting results could be caused by our antibody not recognising the proinsulin produced due to incomplete folding, or sub-optimal treatment of the membrane. Of course there is also the possibility that our strain is not producing proinsulin, although due to the positive results using hrGFP we are confident that our expression system works. Other effects such as mRNA folding or rapid protein degradation may also be factors leading to us not being able to detect proinsulin in the samples. Future efforts could include a fusion of the proinsulin, a reporter (such as GFP) or a purification tag (such as a His-tag) aiding the detection of proinsulin.

Conclusion

In this section we apply the tools made in the molecular toolbox section, for heterologous protein production in Y. lipolytica. This is done by combining two of our own BioBricks, a constitutive transcription factor alpha (TEF1) promoter native to Y. lipolytica (BBa_K2117000) and the hrGFP gene (BBa_K2117003) codon optimized by the algorithm developed by our team. By combining these two parts using A3 assembly and the cloning flow suggested in the molecular tools section, we created the device BBa_K2117005. By confirming the production of hrGFP, we were able to prove that heterologous protein production is possible when our tool pSB1A8YL. Even though efforts to translate this into proinsulin production failed, the modular nature of pSB1A8YL means that it has potential to become a powerful tool when using Y. lipolytica for research or indsutrial purposes.

References

  1. Blazeck, J., Liu, L., Redden, H., & Alper, H. (2011). Tuning Gene Expression in Yarrowia lipolytica by a Hybrid Promoter Approach. Applied and Environmental Microbiology, 77(22), 7905–7914. doi:10.1128/AEM.05763-11
  2. NIDDK
  3. Zaykov, A. N., Mayer, J. P., & DiMarchi, R. D. (2016). Pursuit of a perfect insulin. Nat Rev Drug Discov, 15(6), 425–439. JOUR. Retrieved from http://dx.doi.org/10.1038/nrd.2015.36
  4. Mayer, J. P., Zhang, F., & DiMarchi, R. D. (2007). Insulin structure and function. Biopolymers, 88(5), 687–713. doi:10.1002/bip.20734
  5. Baeshen, N. A., Baeshen, M. N., Sheikh, A., Bora, R. S., Ahmed, M. M. M., Ramadan, H. A. I., … Redwan, E. M. (2014). Cell factories for insulin production. Microbial Cell Factories, 13(1), 141. doi:10.1186/s12934-014-0141-0
  6. IDF annual report 2015

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