Part:BBa_K5182020
Used to examine the UVR8 photocontrol system and can be used as a BioBrick to replace the luciferase
SJTU-BioX-Shanghai 2024
Background
In this composite part, the UAS sequence can be recognized by the Gal4 BD sequence, the luciferase is used to detect the regulatory effect of the light control system, and the P2A sequence is used to promote the translation of the same mRNA strand for the expression of GFP and RUP2, and the expression of RUP2 will play a negative feedback role in the system.
Figure 1. We used RUP2 (BBa_K5182003) with the removal of RUP2 in a set of composite part, and measured the high and low expression of Luciferase reporter gene using enzyme marker
Figure 2. Schematic illustrations of three experimental systems
In the first system, we sought to quantitatively characterize the strength of UV-B-activated gene expression using the Luciferase reporter gene. In the second system, we employed a novel element, RUP2, to construct a negative feedback system, controlling the expression of the reporter gene within an appropriate range.
We performed transfections in HEK293T cells, irradiated with UV-B 24 hours post-transfection, and obtained results after UV-B irradiation.
Figure 3. Schematic diagram of the timeline for the cell experiment process
In terms of experimental design, we used a co-transfection group with the reporter gene plasmid, UVR8-VP64 plasmid, and COP1-NLS-Gal4 plasmid as the experimental group. We set up three control groups containing only the reporter gene plasmid, the reporter gene plasmid + UVR8-VP64 plasmid, and the reporter gene plasmid + COP1-NLS-Gal4 plasmid. This design was intended to rigorously demonstrate that the transcription activation system requires the coordinated function of all gene components, rather than the action of individual components. Additionally, we set up controls between irradiated and non-irradiated UV-B to illustrate the regulatory function of UV-B in the system. The schematic diagram of the transfection experimental design for the experimental and control groups is shown below:
Figure 4. Schematic diagram of the transfection experimental design for the control group cell experiments
For the experiments of systems 2 and 3, we lysed the cells according to the protocol and added firefly luciferase detection reagent, observing chemiluminescence using a plate reader. The statistical results are shown in the figure below:
Firstly, in order to quantitatively characterize the impact of UV-B irradiation on the transcriptional expression of the reporter gene, we conducted two parallel experiments. One set of experiments was subjected to UV-B irradiation, while the other served as a control. After the same duration, we lysed the cells and added a luciferin substrate to measure the activity of luciferase.
Figure 5. Result of Luciferase Reporter Gene Assay With Or Without UV-B Exposure
As illustrated in the figure 7, the impact of UV-B irradiation on the expression of the luciferase reporter gene in the complete system containing three genetic elements is significant. After exposure to UV-B, the expression of the reporter gene increased to nearly five times that of the original, indicating that the gene system we designed is capable of sensitively responding to UV-B input and using the expression of the reporter gene as an output. In other groups containing only two genetic elements or just the reporter gene plasmid, the presence or absence of UV-B does not significantly affect the expression of the reporter gene. This demonstrates that each element in the system is indispensable, and also solidifies the experimental results through the control.
However, we observed that the expression of the target gene was excessively strong after UV-B irradiation, which does not align with the requirement for stable and moderate expression of XPC in practical application scenarios. To further optimize the system, we incorporated the P2A-RUP2 element into the genetic components, constructing a negative feedback regulation system to control the expression level of the reporter gene at a reasonable level under UV-B irradiation.
Figure 8. Luciferase Reporter Gene Assay Analysis of Different Gene Bricks Groups
As shown in the figure 8, the left side represents the performance of the original gene module combination under UV-B irradiation, while the right side represents the performance of the gene combination after the addition of the P2A-RUP2 negative feedback regulation system under UV-B irradiation. It can be observed that the incorporation of the P2A-RUP2 negative feedback regulation system has brought the expression of the reporter gene under control. However, there are issues present, such as the reporter gene expression levels not significantly differing with or without the UVR8-VP64 element. Upon analyzing this issue, we believe that RUP2 competes too strongly with COP1, to the extent that UVR8 can hardly bind with COP1 to enhance transcription initiation after entering the nucleus. To address this issue, we subsequently introduced rare codons into RUP2 in the hope of reducing its expression level, weakening the negative feedback regulation effect, and allowing the reporter gene to express at an appropriate level without being excessively high.
Those Luciferase Reporter Gene Assays provide a quantitative measure of the transcriptional activity of different gene regulatory elements and is a valuable tool for assessing the functionality of gene constructs in synthetic biology applications. It can be seen that we successfully quantitatively determined the activation of reporter gene expression by UV-B and verified that our designed P2A-RUP2 negative feedback regulation system can stably control the expression of the target gene to a reasonable level.
Usage and Biology
We constructed and demonstrated the feasibility of the UV-B-activated reporter gene transcription system. We also quantitatively determined the strength of reporter gene transcription and designed elements to optimize the system, enabling the expression of the target gene to be controlled at a reasonable level. This lays the foundation for applying this expression system to the controllable and stable expression of XPC. Subsequent creators only need to replace the position of BBa_K5182013 in this composite part with the gene they are interested in, and then jointly use our BBa_K5182017, BBa_K5182018 elements to achieve UVB signaling regulation of gene expression.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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