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

“Productivity is never an accident. It is always the result of a commitment to excellence, intelligent planning, and focus efforts” Paul J. Meyer It's your life. Live big.

Introduction

“By 2040, 642 million adults will have diabetes”³
Someone famous in Source Title

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.

We aim to demonstrate this versatility of our Y. lipolytica strain as a cell factory by producing an extracellular heterologous protein. Our first attempt is the production of hrGFP, followed by proinsulin. Moreover we also attempt to produce the metabolic product 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.

hrGFP

Design

In order to test if we were able to clone a of a construct in E. coli pSB1A8YL, which would be expressed, and ultimately prove that we were able to express genes in Y. lipolytica, we chose to develop our own BioBricks. Namely a constitutive TEF promoter (BBa_K2117000) and a codon optimized hrGFP gene (BBa_K2117003) previously used successfully in Y. lipolytica¹. By combining these two parts in a device (BBa_K2117004), the expression should, in theory, be easily detected due to the fluorescence signal produced. The cloning flow can be seen in Figure 8.

Results

The parts were ordered as gBlocks and assembled in E. coli using A3 assembly. The assembly was confirmed using PCR, restriction analysis and sequencing. 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 XX shows the Y. lipolytica PO1f cells under a florescence microscope with 100x magnification.

Proinsulin

Role of insulin in our bodies

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.

Insulin biosynthesis

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 E⁵. The mature insulin contains chains A and B linked by 3 disulphide bonds and in total comprises of 51 amino acids.

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.

Goal

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.

Numbers of diabetes

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.

In 2014 the global insulin market reached $24 billion and it will double by 2020 achieving level of $48 billion.

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.

Design

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. In order to ensure the high rate of transcription we applied the native promoter of Y. lipolytica, TEF1. 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. 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.

Results

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.

Beta-carotene

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.

Numbers of vitamin A

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. 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).

Biosynthesis of the beta-carotene

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-carotene⁶.

Design

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.

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.

Results

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.

Conclusion

TBD

References

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
http://www.who.int/
Guariguata, L., et al. "Global estimates of diabetes prevalence for 2013 and projections for 2035." Diabetes research and clinical practice 103.2 (2014): 137-149.
http://www.idf.org/sites/default/files/IDF_AnnualReport_2015_WEB.pdf
http://www.prnewswire.com/news-releases/global-human-insulin-market-size-of-24-billion-in-2014-to-witness-13-cagr-during-2015---2020-518797291.html
Baeshen, Nabih A., et al. "Cell factories for insulin production." Microbial cell factories 13.1 (2014): 1.
https://2011.igem.org/Team:Johns_Hopkins/Project/VitA
http://www.bccresearch.com/market-research/food-and-beverage/carotenoids-global-market-report-fod025e.html

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-                         <h1>Molecular toolbox<p class="lead">In the substrate section we established that <i>Yarrowia Lipolytica</i> constitutes a great platform utilizing waste streams. In order to unlock the potential of <i>Y. Lipolytica</i>, we developed a molecular toolbox allowing us to efficiently engineering <i>Y. Lipolytica</i>. In this section we present the theory and results of the development of a BioBrick backbone and CRISPR tools for <i>Y. Lipolytica</i></p></h1>
+                         <h1>Products<p class="lead">In the Molecular tools section we developed several molecular tools, potentially allowing us to genetically engineer <i><i>Y. 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>Man is a tool-using animal. Without tools he is nothing, with tools he is all.</p>
+                             <p>“Productivity is never an accident. It is always the result of a commitment to excellence, intelligent planning, and focus efforts”
-                             <small>Thomas Carlyle <cite title="Source Title">Signs of the Times (1829)</cite></small>
+                             <small>Paul J. Meyer <cite title="Source Title">It's your life. Live big.</cite></small>
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-            <h2 class="h2">BioBrick plasmid</h2>
+        <h2 class="h2">Introduction</h2>
-            <h3 class="h3">Introduction</h3>
-            <p>
-                A key part of synthetic biology is to streamline the process of engineering biological systems, by standardizing parts and methods<sup><a href="#references">1</a></sup>. Perhaps the most versatile standards available is the BioBrick standard, in part due to the contributions made during the annual iGEM competition. The BioBrick registry currently has over 20,000<sup><a href="#references">2</a></sup>, and by creating BioBrick plasmid backbones compatible with a new organism, one is effectively unlocking the entire BioBrick registry available for that specific organism. Realizing this, it was decided to develop a plasmid that supports the BioBrick standard and replicates in <i>Y. Lipolytica</i>. Due to the convenience of manipulating <i>Escherichia coli</i> it was determined to develop a shuttle vector that allows for cloning and confirmation of the construct in <i>E. coli</i>, before the construct is transformed in <i>Y. Lipolytica</i>. Additionally, as the only replicative plasmids currently available for <i>Y. Lipolytica</i> is low copy yeast chromosomal plasmids (YCp)<sup><a href="#references">3</a></sup> this allows for high amounts of DNA to easily be propagated in <i>E. coli</i>, before the the plasmid is purified and transformed into <i>Y. Lipolytica</i>. Figure 1 shows a suggested workflow for the proposed BioBrick plasmid.
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-                <figcaption class="figure-caption"><strong>Figure 1:</strong> The suggested workflow using pSB1A8YL described above. The workflow is broken down to three distinct steps: cloning, confirmation and expression. Cloning is the assembly of the construct, which is conducted in <i>E. coli</i> using typical cloning methods. Confirmation is where the identity of the construct is confirmed, using analytical methods such as PCR, analytical restriction enzyme digestion and/or sequencing. Expression includes the work done in the final host organism, <i>Y. lipolytica</i>, and includes inserting the construct and optimization of the expression and cultivation conditions.</figcaption>
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-                <div class="caption">DESCRIPTION</div>
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-        <h3 class="h3">Design</h3>
-            <p>
-            For the design of the plasmid, we decided to incorporate a high copy <i>E. coli</i> part for cloning and propagation DNA. The design was based on the pUC19 vector as it fulfils the criteria of being high copy<sup><a href="#references">4</a></sup>, while perhaps being one of the most widely used cloning vectors for <i>E. coli</i>. To support the BioBrick standard, we only used the ampicillin resistance and replication origin elements of the plasmid. It was found that the sequence in and between these elements did not contain any restriction sites of any current BioBrick assembly standard, thus no further modification of the sequence was needed.
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+                <p>“By 2040, 642 million adults will have diabetes”<sup><a href="#references"> 3</a></sup></p>
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+                 <small>Someone famous in <cite title="Source Title">Source Title</cite></small>
-                    For the <i>Y. Lipolytica</i> part of the plasmid we decided to base the design on the pSL16-CEN1-1(227), as it has found to exhibit high transformation efficiency compared to similar plasmids<sup><a href="#references">5</a></sup>, and perhaps for this reason this plasmid and its derivatives are utilized in many recent studies.<sup><a href="#references">6,7,8</a></sup> Again only the sequence of the replicative and selective elements were chosen. Although, the original sequence was not BioBrick compatible, and thus it was decided to order the sequence as a gBlock. This also introduced the added benefit of being able to incorporate the BioBrick prefix, suffix and a 5’ terminator in the gBlock and exchange the original leucine autotrophy marker with a uracil autotrophy marker allowing for negative selection of the plasmid with 5-Fluoroorotic Acid (5-FOA)<sup><a href="#references">9</a></sup>. In order to comply with the iGEM plasmid nomenclature<sup><a href="#references">10</a></sup>, the plasmid was dubbed “pSB1A8YL”, YL was added in the end to emphasize that the plasmid is used for <i>Y. Lipolytica</i>. Figure 2 shows a graphical representation of the sequence map of pSB1A8YL.
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-                        <figcaption class="figure-caption"><strong>Figure 2:</strong> Sequence map of pSB1A8YL. The colored blocks represents the following: Orange: pUC19 part, Blue modified pSL16-CEN1-1(227) part, pink: BioBrick prefix, purple: BioBrick suffix, red: terminator, green selection markers, grey: origin of replication.  The full annotated sequence can be found <a href="http://parts.igem.org/Part:BBa_K2117009">HERE</a>. </figcaption>
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-                        <figcaption class="figure-caption"><strong>Figure 3:</strong> PCR fragments with USER tails, which were fused to construct pSB1A8YL. The fragment lengths can be seen on the ladder. The primers used and the theretical fragment length can be seen under the bands.</figcaption>
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-                    The pUC19 part and the gBlock fragments was amplified using primers with USER tails, and fused using USER cloning (See Figure 3).
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-            The USER reactions was transformed into chemically competent <i>E. coli</i> DH5alpha cells, and purified. The identity of the product was checked using PCR, restriction analysis (see Figure 4) and sequencing (data not shown).
+				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>Yarrowia lipolytica</i> offers a wide range of substrate tolerance.
+			</p><p>
+				We aim to demonstrate this versatility of our <i>Y. lipolytica</i> strain as a cell factory by producing an extracellular heterologous protein. Our first attempt is the production of hrGFP, followed by proinsulin. Moreover we also attempt to produce the metabolic product 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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-                <img id="img4" class="enlarge img-responsive figure-img" src="https://static.igem.org/mediawiki/2016/7/7d/T--DTU-Denmark--pSB1A8YL_confirmation.png" alt="DESCRIPTION">
-                <figcaption class="figure-caption"><strong>Figure 4:</strong> pSB1A8YL undigested and linearized. Analytical PCRs are also included in the right of the gel picture. The fragment lengths can be seen on the ladder. The restriction enzymes and primers used and the theretical fragment length can be seen under the bands</figcaption>
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+        <h2 class="h2">hrGFP</h2>
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+             <h3 class="h3">Design</h3>
-                <div class="caption">DESCRIPTION</div>
+                <p>
-             </div>
+             In order to test if we were able to clone a of a construct in <i>E. coli</i> pSB1A8YL, which would be expressed, and ultimately prove that we were able to express genes in <i>Y. lipolytica</i>, we chose to develop our own BioBricks. Namely a constitutive TEF promoter (<a href="http://parts.igem.org/Part:BBa_K2117000">BBa_K2117000</a>) and a codon optimized 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>. By combining these two parts in a device (<a href="http://parts.igem.org/Part:BBa_K2117004">BBa_K2117004</a>), the expression should, in theory, be easily detected due to the fluorescence signal produced. The cloning flow can be seen in Figure 8.
-        <h3 class="h3">Testing</h3>
-            <p>
-             After having confirmed the identity of the plasmid, we set out to test its functionality. This was done by: 1. Testing the plasmids replicability and selectivity in <i>Y. Lipolytica</i> and 2. testing the plasmids cloning capabilities in <i>E. coli</i>.
              </p>
-            <h5 class="h5">1. Replicability and selectability in Yarrowia lipolytica</h5>
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-                    The pSB1A8YL plasmid was purified from <i>E. coli</i> DH5alpha, and transformed in <i>Y. Lipolytica</i> PO1f cells. The transformants was selected on selective dropout media not containing uracil, thus only yielding uracil autotroph transformants. A negative control was included substituting the plasmid for MQ water.
-                    The transformations only yielded colonies on the plated containing the cells which were transformed with the plasmid. To ensure that these results indeed meant that our plasmid was stably replicating in the <i>Y. Lipolytica</i> cells, a few colonies were subjected to colony PCR (see Figure 5).
-                    </p>
-                    <p>
-                    These results confirm that pSB1A8YL replicates in <i>Y. Lipolytica</i>, and the chosen uracil selection marker allows for selection of transformants.
-                    To further assess the functionality of pSB1A8YL, the possibility of counter selection was investigated. This was done by growing colonies containing pSB1A8YL on plates containing 5-FOA. Colonies appearing on these plates were then transferred to selective dropout media not containing uracil. As no growth was observed on the latter plate, this proved that pSB1A8YL supports counter selection.
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-                    <img id="img5" class="enlarge img-responsive figure-img" src="https://static.igem.org/mediawiki/2016/thumb/3/34/T--DTU-Denmark--pSB1A8YL_Ylip_cPCR.png/519px-T--DTU-Denmark--pSB1A8YL_Ylip_cPCR.png" alt="DESCRIPTION" width="350">
-                    <figcaption class="figure-caption"><strong>Figure 5:</strong> Colony PCR of pSB1A8YL in <i>Y. lipolytica</i>. The fragment lengths can be seen on the ladder. The primers used and the theoretical fragment length can be seen under the bands</figcaption>
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-<h5 class="h5">2. Cloning capabilities in <i>E. coli</i></h5>
-            <p>
-            In order to test this, we decided to produce a device using BioBricks from the distribution kit which would allow us to easily assess whether the cloning were successful. When deciding BioBricks that would allow this, we received inputs from the SDU iGEM team. We ended up choosing the strong Andersson promoter/strong RBS BioBrick (<a href="http://parts.igem.org/Part:BBa_K880005">BBa_K880005</a>) and pair this with three chromoproteins: amilCP (<a href="http://parts.igem.org/Part:BBa_K592009">BBa_K592009</a>), amilGFP (<a href="http://parts.igem.org/Part:BBa_K592010">BBa_K592010</a>) and mRFP (<a href="http://parts.igem.org/Part:E1010">BBa_E1010</a>), which would allow us to easily pick transformants and visually inspect if the cloning was successful. The cloning flow is shown in Figure 6.
-            </p>
              <figure class="figure">
-             <img id="img6" class="enlarge img-responsive figure-img" src="https://static.igem.org/mediawiki/2016/thumb/c/c7/T--DTU-Denmark--Cloning_flow1.png/800px-T--DTU-Denmark--Cloning_flow1.png" alt="DESCRIPTION">
+             <img id="img9" 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">
-             <figcaption class="figure-caption"><strong>Figure 6:</strong> Cloning flow of the test of pSB1A8YL in <i>E. coli</i>. The expression of the chromoproteins should yield a color ouput detectable by visual inspection.</figcaption>
+             <figcaption class="figure-caption"><strong>Figure 8:</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>
              </figure>
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              <div class="caption">DESCRIPTION</div>
              </div>
+            <h3 class="h3">Results</h3>
              <p>
-             The BioBricks were retrieved from the distribution kit, and assembled with our plasmid as carrier backbone using standard 3A assembly and transformed into chemically competent <i>E. coli</i> DH5alpha cells. The transformants yielded colored colonies (See Figure 7), and the identity of the constructs were confirmed using restriction analysis and PCR (data not shown). These results confirm that pSB1A8YL can be used for cloning in <i>E. coli</i> and supports the BioBrick standard.
+             The parts were ordered as gBlocks and assembled in <i>E. coli</i> using A3 assembly. The assembly was confirmed using PCR, restriction analysis and sequencing. 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 XX shows the <i>Y. lipolytica</i> PO1f cells under a florescence microscope with 100x magnification.
-            </p>
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-            <img id="img7" class="enlarge img-responsive figure-img" src="https://static.igem.org/mediawiki/2016/thumb/8/85/T--DTU-Denmark--Colored_colonies.png/799px-T--DTU-Denmark--Colored_colonies.png" alt="DESCRIPTION">
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-            <img id="img8" class="enlarge img-responsive figure-img" src="https://static.igem.org/mediawiki/2016/thumb/3/3c/T--DTU-Denmark--Color_constructs_restriction_analysis.png/799px-T--DTU-Denmark--Color_constructs_restriction_analysis.png" alt="DESCRIPTION">
-            <figcaption class="figure-caption"><strong>Figure 7:</strong> Actual results from the test of pSB1A8YL in <i>E. coli</i>. Top: A color output was visible both in liquid culutres, pellets and on plates. Bottom: Restriction analysis of the constructs. The fragment lengths can be seen on the ladder. The restriction enzymes used and the theretical fragment length can be seen under the bands</figcaption>
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+            </p>
          </div>
-         <div><a class="anchor" id="section-2"></a>
+         <div><a class="anchor" id="section-3"></a>
-         <h2 class="h2">CRISPR</h2>
+         <h2 class="h2">Proinsulin</h2>
-             <h3 class="h3">Introduction</h3>
+             <h3 class="h3">Role of insulin in our bodies</h3>
                  <p>
-                    By creating a modular plasmid that is compatible with the BioBrick standards and that is able to replicate in both <i>E. coli</i> and <i>Y. lipolytica</i>, the entire biobrick registry has been made available for use in <i>Y. lipolytica</i>. However, one important aspect of industrial-scale protein expression is the stable and homogenous expression of relevant pathway genes. In production organisms with multi-enzyme pathways there is often a need for accurate control of the expression levels of the associated genes, both in terms of promoter strength and copy numbers<sup><a href="#references">11</a></sup>. Plasmid based systems for the expression of heterologous genes are widely used, but offer limited control of copy number and in many cases show notable segregational instability even during growth in selective media. Therefore, it is often preferred to integrate the genetic pathway of interest into the genome of the production organism of choice to ensure long-term strain stability and homogenous expression of pathway genes<sup><a href="#references">11</a></sup>.
+					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.
                  </p>
+            <h3 class="h3">Insulin biosynthesis</h3>
                  <p>
-                     The CRISPR-Cas9 has revolutionized the way in which model organisms can be engineered. In addition, it has made genetic engineering applicable to a wide range of organisms that were previously considered to be uninteresting for cell factory purposes, largely because the genetic engineering tools for these organisms were under characterized. The CRISPR-Cas9 system is ideally suited for the integration of heterologous genes in production organisms, as it relies on Double Strand Break (DSB) induction and its subsequent repair by homologous recombination (HR). In addition, almost any sequence of interest can be targeted with the most commonly used Cas9 from <i>Streptococcus pyogenes</i><sup><a href="#references">12</a></sup>. The basic mechanism of CRISPR-Cas9 is illustrated in Figure 9, for a more detailed overview the reader is referred to Nødvig et al.<sup><a href="#references">12</a></sup>
+                     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<sup><a href="#references">5</a></sup>. The mature insulin contains chains A and B linked by 3 disulphide bonds and in total comprises of 51 amino acids.
+                </p><p>
+                    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.
                  </p>
+             <h3 class="h3">Goal</h3>
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-                <img id="img10" class="enlarge img-responsive figure-img" src="https://static.igem.org/mediawiki/2016/8/8d/T--DTU-Denmark--CRISPRCas9overview.png" alt="DESCRIPTION">
-                <figcaption class="figure-caption"><strong>Figure 9:</strong> Overview of the CRISPR-Cas9 mechanism of DSB induction. The systems consists of two components, the endonuclease Cas9 and a sgRNA. Cas9 is targeted for site-specific DSB induction by the sgRNA. More specifically, a 20 bp region of the sgRNA, known as the protospacer, recognizes the target site through base-pairing. This 20 bp protospacer is easily substituted, allowing for a very modular system. The only restriction for the CRISPR-Cas9 system is the requirement for a Protospacer Adjacent Motif (PAM) in the target region. For the most commonly used Cas9 (from <i>Streptococcus pyogenes</i>) this is the frequently occurring NGG sequence. Reproduced from Nødvig et al.<sup><a href="#references">12</a></sup></figcaption>
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                  <p>
-                     We adapt the CRISPR-Cas9 system for use also in <i>Y. lipolytica</i> in order to obtain a proof-of-concept of the construction of a stable production strain. We do so by a proof-of-concept integration of a gene in <i>Y. lipolytica</i>. In addition, we obtain a proof-of-concept of the deletion of a native <i>Y. lipolytica</i> gene. With these proof-of-concepts, we envision that the metabolic flux towards a possible compound of interest could be increased, leading to tailored <i>Y. lipolytica</i> strains for cell factory engineering purposes.
+                     In order to demonstrate <i>Yarrowia lipolytica</i> as a versatile cell factory we aim to produce proinsulin as an answer to increasing global problem with diabetes.
                  </p>
+             <h3 class="h3">Numbers of diabetes</h3>
-             <h3 class="h3">Experimental design</h3>
                  <p>
-                    The CRISPR-Cas9 system has previously been successfully applied to <i>Y. lipolytica</i> by Schwartz et al.<sup><a href="#references">8</a></sup> As this was a proof-of-concept study, the native <i>Y. lipolytica</i> <i>PEX10</i> gene was targeted for disruption, which allowed for an easily selectable phenotype. This is because <i>PEX10</i> codes for Peroxisome biogenesis factor 10, a protein that is involved in peroxisome biogenesis. Consequently, <i>PEX10</i> disruption results in an inability to catabolize long-chain fatty acids. We set out to confirm the observed <i>PEX10</i> deletion. In addition, we set out to integrate <i>URA3</i>, a commonly used yeast auxotrophic marker gene which codes for Orotidine 5’-phosphate decarboxylase, an enzyme involved in pyrimidine biosynthesis.
+					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) <sup><a href="#references">3</a></sup>.
+                </p><p>
+                    In 2014 the global insulin market reached $24 billion and it will double by 2020 achieving level of $48 billion.
+                </p><p>
+					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.
+				</p>
+            <h3 class="h3">Design</h3>
+                <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.
+					In order to ensure the high rate of transcription we applied the native promoter of <i><i>Y. lipolytica</i></i>, TEF1.
+					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 <i>Y. lipolytica</i> shuttle vector using either 3A assembly, Gibson assembly or USER cloning.
+					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.
                  </p>
+             <h3 class="h3">Results</h3>
                  <p>
-                     We had obtained both a commonly used <i>Y. lipolytica</i> laboratory strain (PO1f, <i>MATA</i> <i>URA3</i>-302 <i>LEU2</i>-270 <i>XPR2</i>-322 <i>AXP2</i> Δ<i>NU49</i> <i>XPR2::SUC2</i>; obtained from Schwartz et al.<sup><a href="#references">8</a></sup>) and also this same strain with a knockout of the Non-Homologous End-Joining (NHEJ) DNA repair pathway gene <i>Ku70</i> (PO1fΔ<i>Ku70</i>, <i>MATA</i> <i>URA3</i>-302 <i>LEU2</i>-270 <i>XPR2</i>-322 <i>AXP2</i> Δ<i>NU49</i> Δ<i>Ku70</i> <i>XPR2::SUC2</i>; obtained from Schwartz et al.<sup><a href="#references">8</a></sup>). The NHEJ pathway for DNA repair is commonly disrupted in laboratory strains to improve the efficiency of CRISPR-Cas9 induced HR integration of heterologous DNA. However, an intact NHEJ pathway can be utilized to disrupt genes without the need for an DNA repair template, as this pathway is intrinsically more error-prone than HR. Thus, in order to disrupt the native <i>Y. lipolytica</i> <i>PEX10</i> gene, we used a CRISPR-Cas9 plasmid (pCRISPRyl) in the PO1f strain. For the <i>URA3</i> insertion, we co-transformed the same CRISPR-Cas9 plasmid and a linearized HR donor plasmid (pIW501) in the PO1fΔ<i>Ku70</i> strain. An overview of our workflow is provided in Figure 10. Overviews of the pCRISPRyl and pIW501 are provided in Figures 11 and 12, respectively.
+                     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.
                  </p>
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-                <img id="img11" class="enlarge img-responsive figure-img" src="https://static.igem.org/mediawiki/2016/c/c0/T--DTU-Denmark--HRNHEJoverview.png" alt="DESCRIPTION">
-                <figcaption class="figure-caption"><strong>Figure 10:</strong> Overview of the experimental design for the proof-of-concept of gene disruption and gene integration using CRISPR-Cas9. Left: transformation of pCRISPRyl should result in gene disruption through error-prone repair by NHEJ (bases shown in yellow). Right: co-transformation of pCRISPRyl and a HR donor plasmid (pIW501) should result in integration of the desired donor gene (bases shown in orange). For plasmid map details, see Figures 11 and 12. Adapted from Nødvig et al.<sup><a href="#references">12</a></sup></figcaption>
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-                    <img id="img12" class="enlarge img-responsive figure-img" src="https://static.igem.org/mediawiki/2016/8/86/T--DTU-Denmark--pcrispryl.png" alt="DESCRIPTION">
-                    <figcaption class="figure-caption"><strong>Figure 11:</strong> Simplified map of pCRISPRyl. Cas9 is under control of a strong constitutive TEF-derived hybrid promoter and the strong CYC1 terminator. Replication origins for use in <i>E. coli</i> (ori) and <i>Y. lipolytica</i> (Cen1-1) & ORI1001) are shown in yellow. Selection markers for use in <i>E. coli</i> (<i>AmpR</i>) and <i>Y. lipolytica</i> (<i>LEU2</i>) are shown in cyan. The synthetic tRNA promoter for sgRNA expression is shown in purple. A novel protospacer can be inserted by linearization of the plasmid with AvrII and subsequent Gibson assembly of a 60 bp fragment containing the desired protospacer into the plasmid<sup><a href="#references">8</a></sup>. Obtained from Addgene (#70007).</figcaption>
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-                    <img id="img13" class="enlarge img-responsive figure-img" src="https://static.igem.org/mediawiki/2016/6/68/T--DTU-Denmark--piw501.png" alt="DESCRIPTION">
-                    <figcaption class="figure-caption"><strong>Figure 12:</strong> Simplified map of pIW501. The HR donor fragment consists of two flanking regions of 1 kb (shown in grey) and the <i>URA3</i> promoter+CDS+terminator (only the CDS is depicted in purple). The flanking regions target the <i>PEX10</i> locus. The replication origin for use in <i>E. coli</i> (ori) is shown in yellow, the selection marker for use in <i>E. coli</i> (<i>AmpR</i>) is depicted in cyan. Prior to transformation in <i>Y. lipolytica</i>, the plasmid is linearized with BamHI in order to increase HR efficiency. Obtained from Schwartz et al.<sup><a href="#references">8</a></sup></figcaption>
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-            <h3 class="h3">Results & Discussion</h3>
-              <h5 class="h5">1. <i>URA3</i> insertion</h5>
-                  <p>
-                      In order to integrate the <i>URA3</i> gene into the genome of the <i>Y. lipolytica</i> PO1fΔ<i>Ku70</i> strain, we first constructed a pCRISPRyl-derived plasmid which contained a protospacer that targets the <i>PEX10</i> locus. This protospacer was used previously for successful disruption of <i>PEX10</i><sup><a href="#references">8</a></sup>. The pCRISPRyl-derived plasmid (pIW357) was constructed through the Gibson assembly of a 60 bp fragment containing the protospacer of interest into the AvrII linearized pCRISPRyl. Subsequently, pIW357 was purified from selected <i>E. coli</i> DH5ɑ transformants and a double restriction analysis was performed with PstI and AvrII (Figure 13). As the purified pIW357 was no longer digested with AvrII, the protospacer had been correctly inserted. In addition, the protospacer insertion was confirmed through Sanger sequencing (data not shown).
-                  </p>
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-                    <img id="img14" class="enlarge img-responsive figure-img" src="https://static.igem.org/mediawiki/2016/f/f8/T--DTU-Denmark--restrictionanalysispstiavrii.png" alt="DESCRIPTION">
-                    <figcaption class="figure-caption"><strong>Figure 13:</strong> Restriction analysis of pCRISPRyl (lanes 2-3) and pIW357 (lanes 5-8) (left to right). Lane 2: pCRISPRyl digested with PstI. Lane 3: pCRISPRyl digested with PstI and AvrII. Lane 5-8: pIW357 digested with PstI and AvrII (4 unique transformants/plasmids). As pIW357 was digested solely by PstI, it was concluded that the Gibson assembly had been successful and the protospacer had been correctly inserted.</figcaption>
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-                        Next, we tested pIW357 in the <i>Y. lipolytica</i> PO1fΔ<i>Ku70</i> strain. In order to account for Cas9 activity of pIW357, we also transformed the pCRISPRyl as a positive control. The results of these transformations are depicted in Figure 14. With an intact NHEJ repair pathway and/or a template for HR, the induced DSB should easily be repaired. However, as this is the PO1fΔ<i>Ku70</i> strain and as there is no HR donor template available, cells die, indicating that the pIW357 is functioning properly. The observed residual growth can be attributed to either mutations in pIW357 (for example in Cas9 or in the the sgRNA/tRNA promoter) or possible repair by the Microhomology-Mediated End-Joining (MMEJ) pathway<sup><a href="#references">14</a></sup>.
-                    </p>
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-                    <img id="img15" class="enlarge img-responsive figure-img" src="https://static.igem.org/mediawiki/2016/1/10/T--DTU-Denmark--testPS.jpg" alt="DESCRIPTION">
-                    <figcaption class="figure-caption"><strong>Figure 14:</strong> Transformation of pIW357 and pCRISPRyl into the <i>Y. lipolytica</i> PO1fΔ<i>Ku70</i> strain. pIW357 (A), pCRISPRyl (B), control (C), plated on SC-leu media.</figcaption>
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-                    <p>
-                        With a functioning pIW357, we then co-transformed pIW357 and pIW501 (linearized) into the <i>Y. lipolytica</i> PO1fΔ<i>Ku70</i> strain (Figure 15). The observation of growth on SC-ura media indicates that the <i>URA3</i> gene had been successfully integrated. Growth was also observed when solely the linearized pIW501 was transformed. In addition, transformants from the co-transformation of pIW357 and pIW501 were screened for <i>URA3</i> integration through gap-check colony PCR (Figure 16). The observed shift in band size between non-pIW501 transformants and pIW501 transformants corresponds to the 1700 bp size of the <i>URA3</i> promoter+CDS+terminator sequence that is present on pIW501.
-                    </p>
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-                        <img id="img16" class="enlarge img-responsive figure-img" src="https://static.igem.org/mediawiki/2016/1/1f/T--DTU-Denmark--cotransformation.jpg" alt="DESCRIPTION">
-                        <figcaption class="figure-caption"><strong>Figure 15:</strong> Co-transformation of pIW357 and linearized pIW501 into the Y. lipolytica PO1fΔKu70 strain. pIW357+linearized pIW501 (A), linearized pIW501 (B), control (C), plated on SC-ura selective media.</figcaption>
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-                        <figure class="figure">
-                        <img id="img17" class="enlarge img-responsive figure-img" src="https://static.igem.org/mediawiki/2016/7/7a/T--DTU-Denmark--colonypcrmodified.png" alt="DESCRIPTION">
-                        <figcaption class="figure-caption"><strong>Figure 16:</strong> Gap-check colony PCR of <i>URA3</i> integration in the <i>PEX10</i> locus of the <i>Y. lipolytica</i> PO1fΔ<i>Ku70</i> strain. The used gap-check primers anneal slightly upstream and downstream of the 1 kb <i>PEX10</i> upstream and downstream flanking regions, respectively. This allows for a direct comparison of the amplicon size between a control transformant and a transformant which should contain the insert in the same PCR reaction. Although only 3 screened co-transformation transformants are depicted in this Figure, an efficiency of 100% was achieved (12/12). Forward primer: TGAGCGAAACGTCATCTACG. Reverse primer: AGCAGTGAAAAGTCGGGCTA.</figcaption>
-                        </figure>
-                        <!-- The Modal with same picture-->
-                        <div id="img17Modal" class="modal">
-                        <span class="close img17">×</span>
-                        <img class="modal-content" id="img17Img">
-                        <div class="caption">DESCRIPTION</div>
-                        </div>
-                        <p>
-                            The pIW501 is linearized prior to transformation in order to increase HR efficiency and in order to reduce the amount of false positive transformants (a gene cannot be expressed from a linear DNA fragment). However, growth was also observed when solely the pIW501 is transformed, indicating that CRISPR-Cas9 DSB induction is not necessarily required for the integration of an auxotrophic marker gene. We did not have time to compare the efficiencies of co-transformation of pIW357 and pIW501 as compared to solely pIW501 transformation. However, it has been shown previously that co-transformation with a CRISPR-Cas9 plasmid increases the HR efficiency<sup><a href="#references">12</a></sup>. We thus hypothesize that the integration of the <i>URA3</i> gene was facilitated by pIW357. We also did not have time to test the integration of a non-marker gene (which would still allow for an easily selectable phenotype such as <i>GFP</i> or <i>lacZ</i>). This might have been a better option, as the efficiency of non-CRISPR-Cas9 induced HR is lower for a non-marker gene. This would have allowed us to more easily distinguish between CRISPR-Cas9-mediated and non-CRISPR-Cas9-mediated gene integration.
-                        </p>
-                   <h5 class="h5">2. <i>PEX10</i> deletion</h5>
-                      <p>
-                      </p>
          </div>
-         <div class="container">
+         <div><a class="anchor" id="section-4"></a>
-<div class="row"> <!--Must sorround both content and sidebar-->
+        <h2 class="h2">Beta-carotene</h2>
-    <div class="col-md-9 col-sm-10 colLeft"> <!-- LEFT -->
-         <div><a class="anchor" id="section-3"></a>
-            <h2 class="h2">Conclusion</h2>
              <p>
-            We successfully developed a shuttle vector that allows the user to harvest the efficiency and accessibility of cloning in <i>E. coli</i>, while still allowing for replication in <i>Y. lipolytica</i>. We extensively tested the plasmid, and showed that it allows for replication, selection in <i>E. coli</i> and replication, selection and even counterselection <i>Y. lipolytica</i>. The plasmid was shown to be compatible with BioBsricks which, to our knowledge, makes it the first tool that allows for the use BioBricks in <i>Y. lipolytica</i>. It is our hope that other actors in iGEM community will use this tool in order harvest the great potentials of <i>Y. lipolytica</i> as a chassis for biorefineries of the future.
+				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.
-            </p><p>
+            </p>
-            As a proof-of-concept, we have shown that we can integrate a gene of interest into <i>Y. lipolytica</i> and also disrupt a native gene of interest. Although in our project we have used a plasmid-based protein expression system this proof-of-concept validates the use of <i>Y. lipolytica</i> for future cell factory engineering purposes. Here, we have focused on the integration of a commonly used auxotrophic marker gene. However, the CRISPR-Cas9 system can be applied for markerless gene integrations<sup><a href="#references">13</a></sup>. Markerless genetic modifications are advantageous as they do not affect cell physiology and allow for iterative genetic modification cycles of the production organism of interest. In addition, this iterative process can be sped up as the modular nature of the CRISPR-Cas9 system makes it ideally suited for simultaneous multiplex genome editing<sup><a href="#references">13,15</a></sup>.
+            <h4 class="h4">Numbers of vitamin A</h4>
-            </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)<sup><a href="#references">7</a></sup>.
+					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).
+                </p>
+            <h3 class="h3">Biosynthesis of the beta-carotene</h3>
+                <p>
+					Beta-carotene is naturally produced by a range of organisms such as plants and fungi, but neither conventional yeast nor <i>Y. lipolytica</i> has the pathway for biosynthesis. Beta-carotene is produced by four enzymatic steps from farnesyl diphosphate (F-PP), which is naturally produced in <i>Y. lipolytica</i>. 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<sup><a href="#references">6</a></sup>.
+                </p>
+            <h3 class="h3">Design</h3>
+                <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.
+				</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>
+            <h3 class="h3">Results</h3>
+                <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.
+                </p>
          </div>
-            <!-- Reference section -->
+        <div><a class="anchor" id="section-5"></a>
-    <div id="ref_sec"><a class="anchor" id="references"></a>
+        	<h2 class="h2">Conclusion</h2>
-    <h2 class="h2">References</h2>
+            	<p>
-    <ol>
+					TBD
-         <li>Shetty, R. P., Endy, D., & Knight, T. F. (2008). Engineering BioBrick vectors from BioBrick parts. Journal of Biological Engineering, 2(1), 5. article. http://doi.org/10.1186/1754-1611-2-5</li>
+            	</p>
-        <li><a href="http://parts.igem.org/Collections">iGEM collections</a></li>
+         </div>
-        <li>Liu, L., Otoupal, P., Pan, A., & Alper, H. S. (2014). Increasing expression level and copy number of a Yarrowia lipolytica plasmid through regulated centromere function. Fems Yeast Research, 14(7), 1124–1127. doi:10.1111/1567-1364.12201</li>
-        <li>Yanisch-Perron, C., Vieira, J., & Messing, J. (1984). Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mpl8 and pUC19 vectors. Gene, 33(33), 103–119. doi:10.1016/0378-1119(85)90120-9</li>
+		<!-- Reference section -->
-        <li>Yamane, T., Sakai, H., Nagahama, K., Ogawa, T., & Matsuoka, M. (2008). Dissection of centromeric DNA from yeast Yarrowia lipolytica and identification of protein-binding site required for plasmid transmission. Journal of Bioscience and Bioengineering, 105(6), 571–578. doi:10.1263/jbb.105.571</li>
+		<div id="ref_sec"><a class="anchor" id="references"></a>
-        <li>Liu, L., Otoupal, P., Pan, A., & Alper, H. S. (2014). Increasing expression level and copy number of a Yarrowia lipolytica plasmid through regulated centromere function. Fems Yeast Research, 14(7), 1124–1127. doi:10.1111/1567-1364.12201</li>
+		    <h2 class="h2">References</h2>
-        <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>
+		    <ol>
-        <li>Schwartz, C. M., Hussain, M. S., Blenner, M., & Wheeldon, I. (2016). Synthetic RNA Polymerase III Promoters Facilitate High-Efficiency CRISPR-Cas9-Mediated Genome Editing in Yarrowia lipolytica. Acs Synthetic Biology, 5(4), 356–359. doi:10.1021/acssynbio.5b00162</li>
+                <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>Sakaguchi, T., Nakajima, K., & Matsuda, Y. (2011). Identification of the UMP Synthase Gene by Establishment of Uracil Auxotrophic Mutants and the Phenotypic Complementation System in the Marine Diatom Phaeodactylum tricornutum. Plant Physiol, 156(1), 78–89.</li>
+		        <li>http://www.who.int/</li>
-        <li><a href="http://parts.igem.org/Help:Plasmid_backbones/Nomenclature">iGEM nomenclature</a></li>
+		        <li>Guariguata, L., et al. "Global estimates of diabetes prevalence for 2013 and projections for 2035." Diabetes research and clinical practice 103.2 (2014): 137-149.</li>
-        <li>Ronda, C., Maury, J., Jakočiu̅nas, T., Baallal Jacobsen, S. A., Germann, S. M., Harrison, S. J., Borodina, I., Keasling, J. D., Jensen, M. K., & Nielsen, A. T. (2015). CrEdit: CRISPR mediated multi-loci gene integration in Saccharomyces cerevisiae. Microbial Cell Factories, 14(1), 97. http://doi.org/10.1186/s12934-015-0288-3</li>
+				<li>http://www.idf.org/sites/default/files/IDF_AnnualReport_2015_WEB.pdf</li>
-        <li>Nødvig, C. S., Nielsen, J. B., Kogle, M. E., & Mortensen, U. H. (2015). A CRISPR-Cas9 system for genetic engineering of filamentous fungi. PLoS ONE, 10(7), 1–18. http://doi.org/10.1371/journal.pone.0133085</li>
+				<li>http://www.prnewswire.com/news-releases/global-human-insulin-market-size-of-24-billion-in-2014-to-witness-13-cagr-during-2015---2020-518797291.html</li>
-        <li>Jessop-Fabre, M. M., Jakočiūnas, T., Stovicek, V., Dai, Z., Jensen, M. K., Keasling, J. D. & Borodina, I. (2016), EasyClone-MarkerFree: A vector toolkit for marker-less integration of genes into Saccharomyces cerevisiae via CRISPR-Cas9. Biotechnology Journal, 11: 1110–1117. doi:10.1002/biot.201600147</li>
+				<li>Baeshen, Nabih A., et al. "Cell factories for insulin production." Microbial cell factories 13.1 (2014): 1.</li>
-        <li>Sakuma, T., Nakade, S., Sakane, Y., Suzuki, K.-I. T., & Yamamoto, T. (2015). MMEJ-assisted gene knock-in using TALENs and CRISPR-Cas9 with the PITCh systems. Nature Protocols, 11(1), 118–133. http://doi.org/10.1038/nprot.2015.140</li>
+				<li>https://2011.igem.org/Team:Johns_Hopkins/Project/VitA</li>
-        <li>Gao, S., Tong, Y., Wen, Z., Zhu, L., Ge, M., Chen, D., Jiang, S., & Yang, S. (2016). Multiplex gene editing of the Yarrowia lipolytica genome using the CRISPR-Cas9 system. Journal of Industrial Microbiology & Biotechnology, 43(8), 1085–1093. http://doi.org/10.1007/s10295-016-1789-8</li>
+				<li>http://www.bccresearch.com/market-research/food-and-beverage/carotenoids-global-market-report-fod025e.html</li>
-    </ol>
+		    </ol>
-    </div>
+		</div>
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      <div class="col-md-3 col-sm-2 colRight" id="scrollspy">
          <ul class="nav" id="sidebar">
-             <li><a href="#section-1">BioBrick plasmid</a></li>
+             <li><a href="#section-2">Introduction</a></li>
-             <li><a href="#section-2">CRISPR</a></li>
+             <li><a href="#section-1">hrGFP</a></li>
-             <li><a href="#section-3">Conclusion</a></li>
+             <li><a href="#section-3">Proinsulin</a></li>
-             <li><a href="#references">References</a></li>
+             <li><a href="#section-4">Beta-carotene</a></li>
+			<li><a href="#section-5">Conclusion</a></li>
+			<li><a href="#references">References</a></li>
          </ul>
      </div> <!-- /RIGHT -->

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

Revision as of 11:41, 19 October 2016

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

Introduction

hrGFP

Design

Results

Proinsulin

Role of insulin in our bodies

Insulin biosynthesis

Goal

Numbers of diabetes

Design

Results

Beta-carotene

Numbers of vitamin A

Biosynthesis of the beta-carotene

Design

Results

Conclusion

References

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