Creative BioMart is now able to provide customized one-step light controlled mammalian protein synthesis system construction service. We are committed to providing customers with high-quality service with the professional technical team and advanced equipment.
In the past decades, chemically regulated gene expression system in mammalian cells has been one of the driving factors for the rapid development of biotechnology, synthetic biology and biomedical research. Although these gene switches allow temporal control of gene expression and are routinely used, they inherently have some disadvantages, such as limited reversibility and poor spatial control, due to the diffusion-based transport of inducers. To overcome these limitations, several light responsive gene expression systems have been developed in the past few years because light allows noninvasive and reversible regulation with high spatio-temporal resolution.
UV-B resistance 8 (UVR8) is an UV-B sensing protein found in Arabidopsis thaliana. UV8 is responsible for sensing ultraviolet rays in the range of 280-315 nm and initiating plant stress response. It is most sensitive at 285nm, close to the lower limit of UVB.
The expression system relies on the two-hybrid principle is one of the most commonly used light regulated mammalian gene expression systems. Here, a transcriptional activation domain is recruited to the DNA sequence upstream of the (minimum) promoter through light controlled protein-protein interaction to induce gene expression.
These systems rely on light induced interactions between protein 1 and protein 2. P1 is fused with DNA binding domain, which specifically interacts with its corresponding DNA binding sequence. P2 is connected to the transcriptional activation domain (TA). The light induced interaction between P1 and P2 recruited TA to the minimum promoter (P min) to induce the expression of GOI.
Based on the system construction protocol we developed and optimized, our basic expression scheme is as follows
| Step | Description |
| Preparation of the plasmid | Two types of high purity plasmids will be prepared |
| Cell inoculation and transfection | HEK-293T cells will be adopted as the host, if there is no special need. The high-purity plasmid encoding target protein and the plasmid encoding UVB light induced gene expression system were transfected into host cells for incubation. |
| UVB light irradiation and sample collection | Once the first transfected gene is expressed (about 1 hour later), the system is sensitive to UVB light in principle. Samples were collected and stored 3, 6, 12 and 24 hours after the start of lighting |
As an alternative to HEK-293T cells, in principle, any other mammalian cells can be used as long as they can be transfected or transduced with the required genetic components. However, it may be necessary to adjust the protocol (e.g., transfection or transduction method, cell culture medium).
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