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.

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 : 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: 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

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.

↑ Mansouri, M. , Strittmatter, T. , & Fussenegger, M. . (2018). Light-controlled mammalian cells and their therapeutic applications in synthetic biology. Advanced Science.
↑ 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_1-1] Mansouri, M. , Strittmatter, T. , & Fussenegger, M. . (2018). Light-controlled mammalian cells and their therapeutic applications in synthetic biology. Advanced Science.

[ref_2-2] Wang, X. , Chen, X. , & Yang, Y. . (2012). Spatiotemporal control of gene expression by a light-switchable transgene system. Nature Methods, 9(3), 266-269.

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