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Team:Freiburg/Content/Project/Overview
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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 more attractive for the iGEM community <a href="https://2014.igem.org/Team:Freiburg/Project/Vision" class="read-more">Read more about | + | <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 more attractive for the iGEM community <a href="https://2014.igem.org/Team:Freiburg/Project/Vision" class="read-more">(Read more about our contribution to iGEM)</a></p> |
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| - | <p>Gene expression under the control of light systems is in general based on a system of two parts: One part is a complex constantly located at a specific DNA sequence, the second part 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 irradiation it switches into the ON state, causing an interaction between both complexes. This leads to the recruitment of DNA polymerase to the target gene and henceforth the gene is expressed. | + | <p>Gene expression under the control of light systems is in general based on a system of two parts: One part is a complex constantly located at a specific DNA sequence, the second part 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 irradiation it switches into the ON state, causing an interaction between both complexes. This leads to the recruitment of DNA polymerase to the target gene and henceforth the gene is expressed.<a href="https://2014.igem.org/Team:Freiburg/Project/The_light_system">Read More about the Light System</a></p> |
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Revision as of 16:58, 17 October 2014
Vision
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.
Project-Overview
Introduction
Gene expression is one of the most complex and tightly controlled processes within eukaryotic cells. Cellular processes crucially depend on precise spatio-temporal coordination of multiple genes.
In order to study these processes, viral vectors can be used to 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. The main obstacles of this technique are the time consuming introduction of genes, limited amount of different light systems and the interference between these.
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.
Fig.1: Project Project-Overview.
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 (Read more about our contribution to iGEM)
Principle
Fig.2: Principle.
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.
Light System
Gene expression under the control of light systems is in general based on a system of two parts: One part is a complex constantly located at a specific DNA sequence, the second part 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 irradiation it switches into the ON state, causing an interaction between both complexes. This leads to the recruitment of DNA polymerase to the target gene and henceforth the gene is expressed.Read More about the Light System
Fig.3a: Light inducible gene expression.
Fig.3b.: Light inducible gene expression.
Viral Vectors And The Receptor
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. Read More about Viral Vectors
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. Read More about the Receptor
Fig.4: Viral vectors and the receptor.
