Team:Freiburg/Content/Project/Overview

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<p>The rapid development of synthetic biology is accompanied by an increased demand for methods that should not only simplify gene delivery, but also provide spatial and temporal control over gene expression.  
<p>The rapid development of synthetic biology is accompanied by an increased demand for methods that should not only simplify gene delivery, 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 viral gene transfer. In our engineered cell line, illumination with light of a defined wavelength leads to the expression of a specific receptor. This receptor serves as an entry point for a viral vector carrying  the genes to be inserted. This combined approach provides a comprehensive picture of the cellular output resulting from different genetic inputs. The gained knowledge can be used to monitor processes of cell development in a new dimension of complexity and ultimately enable facilitated tissue engineering.
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 viral gene transfer. In our engineered cell line, illumination with light of a defined wavelength leads to the expression of a specific receptor. This receptor serves as an entry point for a viral vector carrying  the genes to be inserted. This combined approach provides a comprehensive picture of the cellular output resulting from different genetic inputs. The gained knowledge can be used to monitor processes of cell development in a new dimension of complexity and ultimately enable facilitated tissue engineering.
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  </p><a href="https://2014.igem.org/Team:Freiburg/Project/Vision" class="read-more">Discover the story behind The AcCELLerator</a>
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<a href="https://2014.igem.org/Team:Freiburg/Project/Vision" class="read-more">Read the story behind <strong>The AcCELLerator</strong></a>
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<h1>Overview</h1>
<h2 id="Project-Overview-Introduction">Introduction</h2>
<h2 id="Project-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>
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<p>Gene expression is one of the most complex and tightly controlled processes within eukaryotic cells. Cellular processes crucially depend on precise coordination of multiple genes.</p>
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<p>Thus, in the last years there have been several approaches aiming to provide spatio-temporal control over gene expression, including <em>optogenetic tools</em>, which are to date the method of choice to address 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 <em>viral vectors</em>. 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 <em>blue light inducible gene expression system</em>, which mediates spatio-temporal control and a <em>MuLV based viral vector</em> 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>In order to study these processes, viral vectors can be used to efficiently deliver diverse genetic elements for regulating gene expression in cells. These can include transcription factors, genome editing devices such as the CRISPR system, or other genes of choice. However, often spatio-temporally controlled introduction of genetic elements is necessary, which is not provided by viral vectors.</p>
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<p>Optogenetic tools such as light-responsive gene regulation systems have become more and more prominent over the last few years, enabling high spatio-temporal control over gene expression. </p>
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<p>For the past six months our team developed a novel application, called <strong>The AcCELLerator</strong>, which combines the advantages of the above mentioned techniques. We use a blue light-inducible gene expression system to spatio-temporally control the cargo delivery via viral vectors. This combination 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>In addition, The AcCELLerator enables the fast and easy generation of stable cell lines. Stable cell lines are required for constant and predictable gene expression, as transient introduction of target genes entails the risk of unstable and discontinuous expression levels. To date, viral vectors are commonly used for the generation of stable cell lines. However, most of them demand work under biosafety level 2 conditions. The AcCELLerator, based on a biosafety level 1 viral vector, simplifies the existing time-consuming methods without the need of special safety precautions. Thereby it makes mammalian research  <a href="https://2014.igem.org/Team:Freiburg/Project/Vision" class="read-more">more attractive for the iGEM community.</a></p>
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<p class="header">Fig.1: Project Project-Overview.</p>
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<h2 id="Project-Overview-Principle">Principle</h2>
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<h2 id="Project-Overview-Principle">The AcCELLarator - Optogenetic Control of Viral Gene Delivery</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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<p>Our tool is 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. 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 DNA cargo of the viral vector is easily exchangeable.</p>
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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>Gene expression systems under the control of light are based on two components: One part is constantly located at a specific promoter-associated DNA sequence, the second is fused to a transactivator domain. The system can be in two different states: ON and OFF. In the dark, the system is in the OFF state. Upon illumination, the system is switched ON. The light-induced interaction of both units leads to the recruitment of a DNA polymerase and henceforth the target gene is expressed.<br>
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<a href="https://2014.igem.org/Team:Freiburg/Project/The_light_system">Read More about the Light System</a></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 to VP16, 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. <a href="https://2014.igem.org/Team:Freiburg/Project/The_light_system">Read More about the Light System</a></p>
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<p class="header">Fig.3a: Light inducible gene expression.</p>
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<p class="header">Fig.3b.: Light inducible gene expression.</p>
 
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<h2 id="Project-Overview-Viral-Vectors">Viral Vectors and the Receptor</h2>
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<h2 id="Project-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. </br> <a href="https://2014.igem.org/Team:Freiburg/Project/The_viral_vector">Read More about Viral Vectors</a></p>
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<p>By using viral vectors it is possible to deliver and stably integrate a specific gene cargo within only four days. As the viral recognition site, a specific receptor represents the key component of viral gene delivery. Only if the receptor is present on the cell surface, the virus is able to insert the gene of interest. Otherwise, the virus cannot enter the cells. <br>  
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<a href="https://2014.igem.org/Team:Freiburg/Project/The_viral_vector">Read More about Viral Vectors</a> and <a href="https://2014.igem.org/Team:Freiburg/Project/Receptor">the Receptor</a></p>
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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. </br> <a href="https://2014.igem.org/Team:Freiburg/Project/Receptor">Read More about the Receptor</a></p>
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<p class="header">Fig.4: Viral vectors and the receptor.</p>
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<a href="https://2014.igem.org/Team:Freiburg/Project/The_light_system">Start our Tour and Read more about the Light System</div>
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Latest revision as of 03:23, 18 October 2014

The AcCELLerator

Summary

The rapid development of synthetic biology is accompanied by an increased demand for methods that should not only simplify gene delivery, 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 viral gene transfer. In our engineered cell line, illumination with light of a defined wavelength leads to the expression of a specific receptor. This receptor serves as an entry point for a viral vector carrying the genes to be inserted. This combined approach provides a comprehensive picture of the cellular output resulting from different genetic inputs. The gained knowledge can be used to monitor processes of cell development in a new dimension of complexity and ultimately enable facilitated tissue engineering.

Read the story behind The AcCELLerator

Overview

Introduction

Gene expression is one of the most complex and tightly controlled processes within eukaryotic cells. Cellular processes crucially depend on precise coordination of multiple genes.

In order to study these processes, viral vectors can be used to efficiently deliver diverse genetic elements for regulating gene expression in cells. These can include transcription factors, genome editing devices such as the CRISPR system, or other genes of choice. However, often spatio-temporally controlled introduction of genetic elements is necessary, which is not provided by viral vectors.

Optogenetic tools such as light-responsive gene regulation systems have become more and more prominent over the last few years, enabling high spatio-temporal control over gene expression.


For the past six months our team developed a novel application, called The AcCELLerator, which combines the advantages of the above mentioned techniques. We use a blue light-inducible gene expression system to spatio-temporally control the cargo delivery via viral vectors. This combination facilitates the delivery of variable exogenous genes and provides control over endogenous genes, both with high spatio-temporal resolution.

In addition, The AcCELLerator enables the fast and easy generation of stable cell lines. Stable cell lines are required for constant and predictable gene expression, as transient introduction of target genes entails the risk of unstable and discontinuous expression levels. To date, viral vectors are commonly used for the generation of stable cell lines. However, most of them demand work under biosafety level 2 conditions. The AcCELLerator, based on a biosafety level 1 viral vector, simplifies the existing time-consuming methods without the need of special safety precautions. Thereby it makes mammalian research more attractive for the iGEM community.

The AcCELLarator - Optogenetic Control of Viral Gene Delivery

image/svg+xml light induciblegene expression virus mediatedgene expression receptoron cell surface transduction light inducedreceptor expression illumination viral vector

Our tool is 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. 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 DNA cargo of the viral vector is easily exchangeable.

Light System

Gene expression systems under the control of light are based on two components: One part is constantly located at a specific promoter-associated DNA sequence, the second is fused to a transactivator domain. The system can be in two different states: ON and OFF. In the dark, the system is in the OFF state. Upon illumination, the system is switched ON. The light-induced interaction of both units leads to the recruitment of a DNA polymerase and henceforth the target gene is expressed.
Read More about the Light System

Viral Vectors And The Receptor

By using viral vectors it is possible to deliver and stably integrate a specific gene cargo within only four days. As the viral recognition site, a specific receptor represents the key component of viral gene delivery. Only if the receptor is present on the cell surface, the virus is able to insert the gene of interest. Otherwise, the virus cannot enter the cells.
Read More about Viral Vectors and the Receptor