Difference between revisions of "Part:BBa K2986003"
 
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<partinfo>BBa_K2986003 short</partinfo>
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<partinfo>BBa_K2986003 short</partinfo>  
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Basic part
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===GAVPO===
  
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===Usage and Biology:===
 
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[[File:The_structure_of_GVAPO.png|200px|thumb|left|Figure1.The_structure_of_GVAPO]]
 
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GVAPO is a synthetic light-switch transgene system, the transactivator binds promoters upon blue-light exposure and rapidly initiates transcription of target transgenes in mammalian cells and in mice. this transgene system provides a robust and convenient way to spatiotemporally control gene expression and can be used to manipulate many biological processes in living systems with minimal perturbation. Vivid (VVD), the smallest light-oxygen-voltage (LOV) domain–containing protein, forms a rapidly exchanging dimer upon blue-light activation. This part utilizes the DNA-binding property of a Gal4(65)-VVD fusion protein would be light-switchable, as light should induce dimerization of the fusion protein, enhance binding to the UASG sequence and activate transcription and removing the light should result in gradual dissociation of the dimers, DNA dissociation and inactivation.
 
  
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<h1>Usage and Biology:</h1>
  
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Light is a more feasible factor to switch the particular gene expression compared to chemicals or other factors because it is easy to be modulated spatiotemporally, and quantitatively.<ref name="ref_1">Mansouri, M. , Strittmatter, T. , & Fussenegger, M. . (2018). Light-controlled mammalian cells and their therapeutic applications in synthetic biology. Advanced Science.</ref> X. Wang et al <ref name="ref_2">Wang, X. , Chen, X. , & Yang, Y. . (2012). Spatiotemporal control of gene expression by a light-switchable transgene system. Nature Methods, 9(3), 266-269.</ref> developed the LightON system, which consists of a single chimeric protein '''(GAVPO) that can forms homodimer and bind to its promoter upon exposure to blue light, initiate transcription of the target gene.''' This system is an suitable  regulated gene expression system for our project, as it has low background expression, low toxicity (need weak light for induction), low interference with endogenous proteins or genes and the capacity for temporal and spatial control, and can be easy to manipulate. These characteristics provide us with the capability for gene activation with good spatial, temporal and quantitative control in an easy-to-use system.
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<html>
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<figure>
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    <img src="https://2019.igem.org/wiki/images/b/bb/T--SUSTech_shenzhen--Light_on_system.png" alt="Figure 1. The principle of LightON system" style="width:70%;" />
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    <figcaption>Figure:
  
  
  
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The principle of LightON system</figcaption>
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</figure>
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</html>
  
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[[File:Light-_dependent_activation_of_Fluc_reporters_based_on_GAVP_with_different_mutations_to_enhance_dimerization.png|200px|thumb|left|Figure2.Light-dependent activation of Fluc reporters based on GAVP with different mutations to enhance dimerization ]]
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<h1>Characterization Experiment:</h1>
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Test their light-dependent impact on transcriptional activity of a firefly luciferase (Fluc) reporter driven by Gal4 binding sites upstream of a TATA box after transient transfection in HEK293 cells, illumination with 0.84 W m−2 460 nm peak light from an LED lamp for 22 h and measurement of expression.  
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'''We have done precise characterization on GAVPO's influence in various level of cell regulation, including transcription, translation and downstream protein secretion.
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Transactivators containing the p65 activation domain (GAVP) or the VP16 activation domain (GAVV) both showed marked light-induced reporter gene transcription, but the GAVP transactivator resulted in much greater gene expression under light exposure conditions.
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<html>
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Mutation of Cys108 in VVD to serine blocked light-inducible gene expression as expected13.
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<figure>
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Mutation of Cys71 to valine in VVD is known to enhance the stability of the light-induced VVD dimer14, and based on the crystal structure of VVD13 we hypothesized that mutating Gln56 of VVD to lysine would form a salt bridge with Asp68 of the other VVD protein and additionaly stabilize the dimer. Both dimer-enhancing mutations, C71V and N56K, in the VVD domain decreased reporter gene expression in the dark, whereas the N56K,C71V double mutant (optimized GAVP (GAVPO)) additionally decreased the background gene expression to a minimal level.
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    <img src="https://2019.igem.org/wiki/images/d/df/T--SUSTech_shenzhen--Differnet_level_measurment.png" alt=" Figure  Schematic diagram of 'Multi-level output strategy' " style="width:100%;" />
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So We used GAVPO in all subsequent studies, and we referred to the gene promoter system based on GAVPO as the light-on (LightOn) system.  
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    <figcaption>Figure :Schematic diagram of 'Multi-level output strategy' </figcaption>
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</figure>
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</html>
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==Plasmid construction==
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To characterize the GAVPO function in mammalian cells, we constructed several plasmids. 5xUAS-mRuby-P2A-hGluc and EF1α-GAVPO-Bla. '''The 5xUAS sequence is the binding site of GAVPO protein. '''The reason why we choose hGluc as our target protein is that this protein has low molecular weight and its the detection assay is sensitive. Thus it can be used to mimic the production and secretion process of low molecular weight proteins such as cytokines. Also, we construct a plasmid 5xUAS-mRuby-P2A-hGluc-P2A-IL-10 to test the accuracy of regulation in a more realistic condition. Next, we constructed a plasmid 5xUAS-mRuby-P2A-hGluc-P2A-IL-8. Since IL8 can promote the activation and chemotaxis of neutrophil, it can be detected by observing the migration of neutrophil which is a common process in inflammation and immune reaction.
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<html><figure>
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    <img src="https://2019.igem.org/wiki/images/d/d3/T--SUSTech_shenzhen--Light_on_system_note.png" alt="Figure 2. The schematic diagram of plasmids" style="width:100%;" />
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    <figcaption>Figure: The schematic diagram of plasmids </figcaption>
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</figure></html>
  
  
  
  
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We also test whether Hela cells are successfully transfected after being Illuminated for 24h through observing red fluorescence in Flow Cytometer. FACS data shows that the LightOn system is successfully constructed into HeLa cell.
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<html><figure>
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    <img src="https://2019.igem.org/wiki/images/7/7a/T--SUSTech_Shenzhen--flow.png" alt=" Figure  Schematic diagram of 'Multi-level output strategy' " style="width:100%;" />
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    <figcaption>Figure :Transfection result</figcaption>
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</figure>
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</html>
  
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[[File:Supplementary_Figure_3.jpeg|200px|thumb|right|Figure3.Comparison of induction levels of Fluc reporters driven by GAVV and the conventional CMV driven vectors.]]
 
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The genetically encoded light-responsive transactivator GAVV consists of the Gal4
 
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DNA binding domain, the VVD light sensor and VP16 transactivation domain.
 
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Experimental conditions were as described for Figure.2. The data were normalized to
 
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the Fluc expression level of the pcDNA3.1-Fluc vector under dark condition. Error
 
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bars, mean ± s.e.m. (n = 4) from the same experiment.
 
  
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After transfection, we need to know the '''optimal condition''' of blue light exposure for GAVPO protein, then we can precisely decide the range of light condition in latter experiments. We designed a series gradients of illumination time and light intensity to set different illumination conditions. Then we obtained the relationship between target protein (interleukin-10) secretion level and light intensity/time to characteize the GAVPO function.
  
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<hr>
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&emsp;(i): '''Relationship between IL-10 expression and light intensity''':
  
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===Design Notes===
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<html>
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How can we coupled this light-switch system with gene coded for the cytokines, which may stay in the cytosol or secrete to the outside of the cells. Whether changing another kind of cells can affect the effect of this system.
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<figure>
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    <img src="https://2019.igem.org/wiki/images/c/cc/T--SUSTech_shenzhen--Light_gradient_data_example.png" "alt="" style="width:100%;" />
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    <figcaption>Figure : Intensity gradient</figcaption>
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</figure>
  
  
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===Source===
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<figure>
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    <img src="https://2019.igem.org/wiki/images/d/d8/T--SUSTech_Shenzhen--result02.png" "alt="" style="width:100%;" />
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    <figcaption>Figure : Illuminating hela-5xUAS-mRuby-P2A-IL10 for 48h with different intensities. a. Measuring the concentration of IL10 by Elisa; b. Measuring red fluorescent with flow cytometer.</figcaption>
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</figure>
  
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This light-switch transgene system is form the lab of Xue Wang, Xianjun Chen & Yi Yang, they published Spatiotemporal control of gene expression by a light-switchable transgene system in Nature Methods 2012.
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&emsp;(ii): '''Relationship between target gene expression and illumination time''':
  
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===SequenceAndFeatures===
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<html>
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<figure>
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    <img src="https://2019.igem.org/wiki/images/2/26/T--SUSTech_shenzhen--Time_gradient_data_example.png" "alt="" style="width:100%;" />
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    <figcaption>Figure 6. Time gradient</figcaption>
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</figure>
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<figure>
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    <img src="https://2019.igem.org/wiki/images/9/94/T--SUSTech_Shenzhen--result04.png" "alt="" style="width:100%;" />
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    <figcaption>Figure: Illuminating hela-5xUAS-mRuby-P2A-IL10 for 60h under 102.4uw blue light. a. Measuring the concentration of IL10 with Elisa; b. Measuring red fluorescent with flow cytometer.</figcaption>
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</figure>
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</html>
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Then, we did characterization of the whole expression process at different levels (transcription, translation, and secretion)<br/>
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After obtaining the optimal condition of illumination, we are able to efficiently quantitatively characterize the whole expression process at different levels. We characterized the transcription process by testing the change of RNA through quantitative PCR. Next, we characterized the translation process by testing the dynamic change of mRuby through flow cytometer. The final step is to characterize the secretion process. Since we have chosen hGluc as our target product, we did it by measuring the chemiluminescence value.<br/>
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[[File:T--SUSTech--yong11.png|550px|thumb|center|Figure5. Result of qPCR test on transcription characterization]]
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[[File:T--SUSTech--yong12.png|550px|thumb|center|Figure6. Result of mRuby flow cytometry test for translation characterization and cell number at each time point]]
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[[File:T--SUSTech--yong13.png|400px|thumb|center|Figure7. Result of hGluc chemiluminescence value on secretion characterization]]
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1. For the qPCR test, RNA has shorter half-life, thus its change is more dynamic compared to secreted protein. Hence, it provide some characteristics of RNA dynamics for further modeling. <br/>
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2. For the flow cytometry test, we obtained characterization data of translation process. <br/>
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3. For the hGluc chemiluminescence test, we characterized the secretion process after protein translation. This set of data enable us to characterize he relationship between light exposure and Gene expression on multi-level (transcription level, translation level and secretion level), which is vital for further acquisition of experimental parameters and model constructions<br/>
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<h1>Source</h1>
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Wang, X. , Chen, X. , & Yang, Y. . (2012). Spatiotemporal control of gene expression by a light-switchable transgene system. Nature Methods, 9(3), 266-269.
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<h1>Sequence and Features</h1>
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<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K2986003 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K2986003 SequenceAndFeatures</partinfo>
  
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===References===
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<h1>References</h1>
 
Wang X , Chen X , Yang Y . Spatiotemporal control of gene expression by a light-switchable transgene system[J]. Nature Methods, 2012, 9(3):266-269.
 
Wang X , Chen X , Yang Y . Spatiotemporal control of gene expression by a light-switchable transgene system[J]. Nature Methods, 2012, 9(3):266-269.

Latest revision as of 03:58, 22 October 2019

light-switchable transactivator

GAVPO

Usage and Biology:

Light is a more feasible factor to switch the particular gene expression compared to chemicals or other factors because it is easy to be modulated spatiotemporally, and quantitatively.[1] X. Wang et al [2] developed the LightON system, which consists of a single chimeric protein (GAVPO) that can forms homodimer and bind to its promoter upon exposure to blue light, initiate transcription of the target gene. This system is an suitable regulated gene expression system for our project, as it has low background expression, low toxicity (need weak light for induction), low interference with endogenous proteins or genes and the capacity for temporal and spatial control, and can be easy to manipulate. These characteristics provide us with the capability for gene activation with good spatial, temporal and quantitative control in an easy-to-use system.

Figure 1. The principle of LightON system
Figure: The principle of LightON system

Characterization Experiment:

We have done precise characterization on GAVPO's influence in various level of cell regulation, including transcription, translation and downstream protein secretion.

Figure Schematic diagram of 'Multi-level output strategy'
Figure :Schematic diagram of 'Multi-level output strategy'

Plasmid construction

To characterize the GAVPO function in mammalian cells, we constructed several plasmids. 5xUAS-mRuby-P2A-hGluc and EF1α-GAVPO-Bla. The 5xUAS sequence is the binding site of GAVPO protein. The reason why we choose hGluc as our target protein is that this protein has low molecular weight and its the detection assay is sensitive. Thus it can be used to mimic the production and secretion process of low molecular weight proteins such as cytokines. Also, we construct a plasmid 5xUAS-mRuby-P2A-hGluc-P2A-IL-10 to test the accuracy of regulation in a more realistic condition. Next, we constructed a plasmid 5xUAS-mRuby-P2A-hGluc-P2A-IL-8. Since IL8 can promote the activation and chemotaxis of neutrophil, it can be detected by observing the migration of neutrophil which is a common process in inflammation and immune reaction.

Figure 2. The schematic diagram of plasmids
Figure: The schematic diagram of plasmids



We also test whether Hela cells are successfully transfected after being Illuminated for 24h through observing red fluorescence in Flow Cytometer. FACS data shows that the LightOn system is successfully constructed into HeLa cell.

Figure Schematic diagram of 'Multi-level output strategy'
Figure :Transfection result


After transfection, we need to know the optimal condition of blue light exposure for GAVPO protein, then we can precisely decide the range of light condition in latter experiments. We designed a series gradients of illumination time and light intensity to set different illumination conditions. Then we obtained the relationship between target protein (interleukin-10) secretion level and light intensity/time to characteize the GAVPO function.

 (i): Relationship between IL-10 expression and light intensity:

Figure : Intensity gradient
Figure : Illuminating hela-5xUAS-mRuby-P2A-IL10 for 48h with different intensities. a. Measuring the concentration of IL10 by Elisa; b. Measuring red fluorescent with flow cytometer.
 (ii): '''Relationship between target gene expression and illumination time''':
Figure 6. Time gradient
Figure: Illuminating hela-5xUAS-mRuby-P2A-IL10 for 60h under 102.4uw blue light. a. Measuring the concentration of IL10 with Elisa; b. Measuring red fluorescent with flow cytometer.

Then, we did characterization of the whole expression process at different levels (transcription, translation, and secretion)
After obtaining the optimal condition of illumination, we are able to efficiently quantitatively characterize the whole expression process at different levels. We characterized the transcription process by testing the change of RNA through quantitative PCR. Next, we characterized the translation process by testing the dynamic change of mRuby through flow cytometer. The final step is to characterize the secretion process. Since we have chosen hGluc as our target product, we did it by measuring the chemiluminescence value.

Figure5. Result of qPCR test on transcription characterization
Figure6. Result of mRuby flow cytometry test for translation characterization and cell number at each time point
Figure7. Result of hGluc chemiluminescence value on secretion characterization

1. For the qPCR test, RNA has shorter half-life, thus its change is more dynamic compared to secreted protein. Hence, it provide some characteristics of RNA dynamics for further modeling.
2. For the flow cytometry test, we obtained characterization data of translation process.
3. For the hGluc chemiluminescence test, we characterized the secretion process after protein translation. This set of data enable us to characterize he relationship between light exposure and Gene expression on multi-level (transcription level, translation level and secretion level), which is vital for further acquisition of experimental parameters and model constructions


Source

Wang, X. , Chen, X. , & Yang, Y. . (2012). Spatiotemporal control of gene expression by a light-switchable transgene system. Nature Methods, 9(3), 266-269.


Sequence and Features

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


References

Wang X , Chen X , Yang Y . Spatiotemporal control of gene expression by a light-switchable transgene system[J]. Nature Methods, 2012, 9(3):266-269.
  1. ↑ Mansouri, M. , Strittmatter, T. , & Fussenegger, M. . (2018). Light-controlled mammalian cells and their therapeutic applications in synthetic biology. Advanced Science.
  2. ↑ Wang, X. , Chen, X. , & Yang, Y. . (2012). Spatiotemporal control of gene expression by a light-switchable transgene system. Nature Methods, 9(3), 266-269.