Team:Freiburg/Content/Project/Overview

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<h1><strong>Vision</strong></h1> <!-- zu bootstrap hinzufügen-->

<p>The rapid development of synthetic biology is accompanied by an increased demand for methods that should not only enable the delivery of various genes, but also provide spatial and temporal control over gene expression. We, the iGEM team Freiburg 2014, designed a system which combines the advantages of two biotechnological approaches – the spatio-temporal resolution of optogenetics and the simplicity of gene transfer offered by viruses. In our engineered cell line light induction leads to the expression of a specific receptor serving as the entry point for the viral vector that carries the genes to be inserted. Hereby the method leads to a comprehensive picture of the cellular output resulting from different genetic inputs. The gained knowledge can be used to monitor the processes of tissue development in a new dimension of complexity and ultimately to engineer tissues. </p><a href="https://static.igem.org/mediawiki/2014/3/3a/TeamFreiburgHomeSlide2.jpg" class="read-more">Read More</a>

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<h1>Overview</h1>
<h2 id="Overview-Introduction">Introduction</h2>
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			 	<p>Gene expression is one of the most complex and tightly controlled processes within eukaryotic cells. Cellular fate and indeed the survival of entire organisms crucially depend on precise spatio-temporal coordination of a multitude of genes.</p>
<p>Thus, in the last years there have been several approaches aiming to provide spatio-temporal control over gene expression, including &nbsp;<strong>optogenetic</strong> <strong>tools</strong>, which are to date the method of choice to adress these challenges. The main obstacles of this technique are the time consuming introduction of genes, limited amount of different light systems and the interference between these. This restricts optogenetic methods regarding the variability and number of genes to be inserted and controlled and makes the procedure time-consuming.</p><p>In contrast, a large variety of different genes can easily be packed and delivered by <strong>viral vectors</strong>. However, gene delivery with viral vectors lacks the ability to target specific subpopulations of cells. For the past six months our team developed a novel application, called The AcCELLerator, which combines the advantages of both approaches. Therefore, we use a <strong>blue light inducible gene expression system</strong>, which mediates spatio-temporal control and a <strong>MuLV based viral vector</strong> that provides an easy way to insert any gene cargo. This combination allows to overcome the drawbacks of both approaches: it facilitates the delivery of variable exogenous genes and provides control over endogenous genes, both with high spatio-temporal resolution.</p>
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			<p class="header">Fig.1: Project overview.</p>
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<h2 id="Overview-Principle">Principle</h2>
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			<p class="header">Fig.2: Principle.</p>
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			<p>Our tool is basically a two-component system, combing specificity of optogenetics with variability of viral vectors. The light system is stably integrated into a cell line. It controls the expression of a certain receptor, CAT1 (cationic amino acid transporter 1). When illuminated with blue light the light system switches into the “ON”-state and the receptor is expressed. Cells expressing the receptor can be recognized by our second component, a viral vector. The cargo of the viral vector is easily exchangeable. The viral vector can integrate any gene of interest stably in the host genome.</p>
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<h2 id="Overview-Light-System">Light system</h2>
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			<p>Light induced expression of target genes bases on a system that consists of mainly two parts: One, a complex of LOV2 fused to Gal4DBD constantly located &nbsp;at a specific DNA sequence, the Gal4UAS. While in the dark, J&alpha; chain is not exposed, therefore, the ePDZ-VP-16 domain can not be recruited and no gene expression is detectable.</p>
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			<p>Upon illumination, the J&alpha; chain of the LOV2-domain becomes accessible enabling the second part of the light system, epdZ fused toVP16, to bind to the J&alpha; chain. The VP-16 domain of the second part acts as a transactivator of transcription which recruits DNA polymerase to the target gene.</p><a href="https://2014.igem.org/Team:Freiburg/Project/The_light_system">Read More</a>
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		  <p class="header"> Fig. 3 LOV2: Light inducible expression system</p>
		  <p class="desc">A: Light system in OFF-state; B: activated light system in ON-state</p>
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<h2 id="Overview-Viral-Vectors">Viral vectors and the receptor</h2>
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			<p>Viral vectors constitute a fast and easy method for gene delivery. By using viral vectors it is possible to deliver and stably express a certain gene cargo within only four days. In order to ensure safety and to enable the insertion of large gene cargos, essential viral genes are transferred to a so-called packaging cell line. This cell line can produce infectious viral particles easily, if it is transfected with a transfer vector carrying the gene of interest.</p><a href="https://2014.igem.org/Team:Freiburg/Project/The_viral_vector">Read More</a>
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			<p>The receptor represents the key component of the AcCELLerator that is essential for the combination of viral vectors and light induced expression systems: Only, if the receptor is present on cells, the virus is able to infect them and insert the gene of interest. Otherwise, the virus can not enter the cell.</p><a href="https://2014.igem.org/Team:Freiburg/Project/Receptor">Read More</a>
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					<p class="header">Fig.4: Viral vectors and the receptor.</p>
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