Difference between revisions of "Part:BBa K2986003"
 
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<partinfo>BBa_K2986003 short</partinfo>  
 
<partinfo>BBa_K2986003 short</partinfo>  
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===GVAPO===  
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===GAVPO===  
  
  
 
<h1>Usage and Biology:</h1>
 
<h1>Usage and Biology:</h1>
  
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GVAPO is a light-switch transgene system, it can act as sensor of blue-light, with the activation of blue light, it can bind to promoter and initiates transcription of upstream gene in a short time. GVAPO is also called a light-switchable transactivator due to its robust and convenient way to spatiotemporally control gene expression.(Xue Wang, 2012). GVAPO explores a new method to control the small molecular inducers, which may diffuse freely and have difficulty to move. Through this way, GVAPO overcome the dilemma happened in chemically regulated gene expression systems. Using blue light is not the first approach for scientists trying to control gene expression with light. Some of them developed UV light activate system to active the caged transactivator or chemical inducer. Infrared laser light was also a tool to induce the target gene expression by heat shock. However, both the UV light and infrared laser need complex equipment, the blue light with GVAPO is relatively a simple and stable way to directly control gene expression process by regulating the photosensitive transactivator.
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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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[[File:T--SUSTech-GVAPO.png|400px|thumb|center|Figure1.GVAPO in mammalian lentivirus expression vector ]]
 
  
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The principle of LightON system</figcaption>
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</figure>
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</html>
  
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We wanted to rationally design a blue-light controlling system in mammalian cells, and the first step is to equip the GVAPO inside the target cells. We designed a plasmid to stably express GVAPO using mammalian lentivirus expression vector. <br/>
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<h1>Characterization Experiment:</h1>
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===Location of features===
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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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5’ LTR (5’ long terminal repeat):1-635 <br/>
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<html>
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PBS (primer binding site): 636-653 <br/>
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<figure>
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Packaging signal: 685-822 <br/>
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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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REE (rev-response element)：1303-1536 <br/>
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    <figcaption>Figure :Schematic diagram of 'Multi-level output strategy' </figcaption>
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cPPT/CTS (central polypurine tract/central termination sequence):2028-2151 <br/>
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</figure>
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EF1A（human elongation factor 1 alpha promoter）：2185-3561 <br/>
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</html>
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GVAPO：3562-5085 <br/>
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==Plasmid construction==
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Blastincdin (encodes the enzyme beta-lactamase, which breaks down the antibiotic ampicillin): 5266-5661 <br/>
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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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WPRE (woodchuck hepatitis virus posttranscription regulatory element):5675-6266 <br/>
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<html><figure>
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3’LTR (3’ long terminal repeat): 6469-7105 <br/>
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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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pUC origin of replication: 7574-8247 <br/>
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    <figcaption>Figure: The schematic diagram of plasmids </figcaption>
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Amp^r (ampicillin resistance gene): 8392-9388 <br/>
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</figure></html>
  
  
  
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Later we transfected this plasmid into Hela cells, we use flow cytometry to test whether we successfully transfected. We found from the result that the peak of the group of cells with blue light exposure horizontally move to the right, compared with the cells under dark conditions. This showed that GVAPO was transfected into Hela cells, and worked as a control the expression of fluorescent using blue light as a switch.
 
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[[File:T--SUSTech-flow cyto.png|450px|thumb|center|Figure2.result of flow cytometry]]
 
  
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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>
 +
    <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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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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<h1>Properties</h1>
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&emsp;(i): '''Relationship between IL-10 expression and light intensity''':
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Although blue light far more less toxic than UV light, long time exposure under blue light can still bring harm to cell growth. Under long-lasting light condition, the reactive oxygen species (ROS) inside the cells will increase, which may damage the nuclear genome, cause mutation on mitochondria DNA and active cell apoptosis. What’s more, the effect taken by the blue light is different with light exposure time and blue light intensity.
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<html>
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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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[[File:T--SUSTech--guangzhao qiangdu.png|550px|thumb|center|Figure3. the form of light intensity gradient]]
 
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[[File:T--SUSTech- guangz shijian.png|550px|thumb|center|Figure4. the form of exposure time gradient]]
 
  
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<figure>
 +
    <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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To found the optimal exposure time and intensity and ensured a good environment for target gene expression as much as possible, we designed a series of time and intensity gradient. We chose the longest exposure time as 60 hours, and set the interval of the gradient to 5 hours, totally we have 12 groups to help us find the best exposure time. As for the intensity gradient, we set the intensity from 0.4μW to 819.2μW, and we also divided them into 12 groups. Then we done Elisa to test the expression of the target gene expression under various gradients, and finally we found that the best light intensity to maximum gene-expression in engineered cell line is 102.4μW and the maximum gene-expression light exposure time for engineered cell line is around 45h.
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&emsp;(ii): '''Relationship between target gene expression and illumination time''':
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[[File:T--SUSTech QIANGDU.png|350px|thumb|center|Figure4.gene expression under intensity gradient (cytokine 10 is target gene, hGluc is a fluorescent to indicate the expression of target gene, the maximum gene-expression light intensity for engineered cell line is 102.4μW)]]
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<html>
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<figure>
 +
    <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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[[File:T--SUSTech-TIME.png|350px|thumb|center|Figure5.gene expression under exposure time gradient (cytokine 10 is target gene, hGluc is a fluorescent to indicate the expression of target gene, the maximum gene-expression light exposure time for engineered cell line is around 45h)]]
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<figure>
 +
    <img src="https://2019.igem.org/wiki/images/9/94/T--SUSTech_Shenzhen--result04.png" "alt="" style="width:100%;" />
 +
    <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>
 +
</html>
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 +
Then, we did characterization of the whole expression process at different levels (transcription, translation, and secretion)<br/>
 +
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/>
 +
 
 +
[[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]]
 +
 
 +
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/>
 +
2. For the flow cytometry test, we obtained characterization data of translation process. <br/>
 +
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/>
 +
 
  
 
<h1>Source</h1>
 
<h1>Source</h1>
  
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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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Wang, X. , Chen, X. , & Yang, Y. . (2012). Spatiotemporal control of gene expression by a light-switchable transgene system. Nature Methods, 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.