Revision as of 16:30, 20 October 2019

light-switchable transactivator

GVAPO

Usage and Biology:

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 robustly initiates transcription of upstream gene in a short time. GAVPO is also called a light-switchable transactivator due to its robust and convenient way to spatiotemporally control gene expression.(Xue Wang, 2012). GAVPO does the small molecular inducers, which may diffuse freely and have hard to control. Through this way, GAVPO 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.

Characterization Experiment:

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.

Figure1.GVAPO in mammalian lentivirus expression vector

5’ LTR (5’ long terminal repeat):1-635
PBS (primer binding site): 636-653
Packaging signal: 685-822
REE (rev-response element)：1303-1536
cPPT/CTS (central polypurine tract/central termination sequence):2028-2151
EF1A（human elongation factor 1 alpha promoter）：2185-3561
GVAPO：3562-5085
Blastincdin (encodes the enzyme beta-lactamase, which breaks down the antibiotic ampicillin): 5266-5661
WPRE (woodchuck hepatitis virus posttranscription regulatory element):5675-6266
3’LTR (3’ long terminal repeat): 6469-7105
pUC origin of replication: 7574-8247
Amp^r (ampicillin resistance gene): 8392-9388

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.

Figure2.result of flow cytometry

After tranfected the GAVPO sequence into HeLa cells, we have done accureate characterization in different scales such as its influence on gene transcription, protein translation and secretion. 1. Determination of optimal illumination conditions
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.

Figure3. the form of light intensity gradient

Figure4. the form of exposure time gradient

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. From the characterization data, 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.

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)

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)

2.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

This GAVPO protein is designed byXue Wang, Xianjun Chen & Yi Yang et al from ECUST, 2012

Sequence and Features

Sequence and Features

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.