Difference between revisions of "Part:BBa K5398631"

Revision as of 10:07, 30 September 2024

Use RAT terminator sequence to improve the leakage BBa_K3739064 system

This part is to lower the leakage level of the BBa_K3739064. Aiming to control the leakage issue, we insert the RAT terminator sequence into the regions between the pBlind promoter sequence and superfolder(sfGFP) reporter gene sequence. Besides, the expression level of LicV is high enough to ensure that the sfGFP can transcript normally due to the strong promoter J23104.

Introduction

EL222 is a natural photosensitive DNA-binding protein that dimerizes and binds DNA upon blue light exposure. It is composed of a N-terminal light-oxygen-voltage(LOV) domain and a C-terminal helix-turn-helix(HTH) DNA-binding domain characteristic of LuxR-type DNA-binding proteins. EL222 could act as a transcriptional activator in a tunable blue light-inducible promoter system. pBlind, a synthetic blue light-inducible promoter, is formed by replacing the lux box which is a 20-bp inverted repeat from the luxI promoter with the 18-bp EL222 binding protein region. When exposed to blue light, EL222 dimerizes and overlaps the -35 region of the promoter thus recuiting RNAP. But a high leakage level of this blue light-induced system is witnessed. Aiming to lower the leakage, XMU-China 2021 replaced the strong promoter BBa_J23106 with pBlind. Although the leakage issue has improved somewhat, a quantity of reporter genes will still be expressed without blue light.

Fig. 1 | EL222 system designed by XMU-China 2021.

The RAT element is an RNA antiterminator sequence that prevents the formation of terminator structures by blocking the creation of hairpin loops in the mRNA, which would normally signal transcription termination. When LicV protein is activated by blue light, it binds to the RAT sequence, stabilizing the transcription process and ensuring that the target gene (sfGFP) is expressed only as needed, achieving precise gene regulation. This design effectively reduces leakage under dark conditions.

Fig. 2 | Improved EL222 system with LicV and RAT.

Usage and Biology

The EL222-based blue light-inducible system allows precise control of gene expression by dimerizing and binding DNA upon blue light exposure. To reduce system leakage, a LicV fusion protein and RAT sequence were introduced, enhancing control by preventing premature transcription termination. This system is ideal for applications requiring tight regulation of gene expression in response to light, such as biosensors and metabolic engineering.

Characterization

Experimental Design

To characterize the improved EL222 blue light-inducible system and evaluate the reduction in leakage, we designed a set of experiments involving four distinct plasmid constructs. Each construct either contains or lacks the RAT terminator sequence and the LicV fusion protein gene. These plasmids were co-transformed into E.coli BL21(DE3) cells, and the fluorescence intensity of the reporter gene (sfGFP) was measured over time under blue light or dark conditions.

The experimental group and three control groups were set up as follows:

①Experimental group (RAT+ LicV+): Contains both the RAT terminator sequence and LicV fusion protein, expected to show minimal leakage in the absence of blue light and induced sfGFP expression under blue light.

②Control group 1 (RAT+ LicV-): Contains the RAT terminator sequence but lacks LicV. This group is expected to exhibit minimal sfGFP expression under both light and dark conditions due to the absence of LicV activation.

③Control group 2 (RAT- LicV+): Contains LicV but lacks the RAT terminator sequence. This group may show increased sfGFP expression and potential leakage in the absence of blue light due to the lack of RNA-based regulation.

④Control group 3 (RAT- LicV-): Lacks both the RAT terminator sequence and LicV. This group is expected to have the highest leakage due to the absence of both regulatory elements.

All four groups were subjected to both blue light exposure and dark conditions during cultivation, leading to a total of eight experimental setups. Bacteria were cultured at 37°C, and samples were taken every two hours over a 16-hour period for fluorescence and OD₆₀₀ measurements by PerkinElmer Ensight multimode plate reader.

Result

The fluorescence intensity of sfGFP was measured at multiple time points across the 16-hour period under both blue light and dark conditions, and the results revealed distinct patterns in gene expression across the different groups. Under dark conditions, the experimental group (RAT+ LicV+) exhibited significantly lower fluorescence compared to Control Group 3 (RAT- LicV-), indicating that the inclusion of the RAT element effectively reduced system leakage in the absence of light. This reduction in fluorescence highlights the success of the RAT element and LicV protein in suppressing background expression when blue light is not present.

Control Group 1 (RAT+ LicV-) displayed consistently low fluorescence under both light and dark conditions, demonstrating that the RAT element alone was sufficient to prevent gene expression, regardless of illumination. This suggests that without the presence of LicV, the downstream gene expression remained effectively inhibited. In contrast, Control Group 2 (RAT- LicV+) showed fluorescence levels similar to those of Control Group 3 under both light and dark conditions, indicating that, in the absence of the RAT sequence, LicV was unable to perform its regulatory function. As a result, gene expression remained uncontrolled, leading to similar fluorescence levels as observed in the fully unregulated system of Control Group 3.

Furthermore, both the experimental group and Control Group 2 exhibited lower fluorescence than Control Group 3 under blue light, suggesting that the introduction of additional regulatory elements, such as the RAT terminator and the LicV protein, may have imposed a metabolic burden that impacted the efficiency of gene expression. Despite this burden, the significant reduction in fluorescence under dark conditions for the experimental group demonstrates that our modifications successfully reduced the background expression, thereby enhancing the overall controllability of the system.

Fig. 3 | Fluorescence intensity over time for all groups.

The microscopy images visually confirm the trends observed in the enzyme-linked measurements. Groups with lower fluorescence intensities in the plate reader data (e.g., Control Group 1) also show weaker fluorescence under the microscope, further supporting the hypothesis that the metabolic burden from the regulatory elements affects sfGFP expression. Conversely, Control Group 3 exhibits stronger fluorescence, consistent with the data from the microplate reader.

Fig. 4 | Fluorescence microscopy images comparing the final time-point samples from different groups.

More information about the project for which the part was created: SAMUS (NAU-CHINA 2024)

Sequence and Features

Reference

[1] Premkumar Jayaraman, Kavya Devarajan, Tze Kwang Chua, et al. Blue light-mediated transcriptional activation and repression of gene expression in bacteria[J]. Nucleic Acids Research, 2016, 44(14): 6994-7005.

[2] Renmei Liu, Jing Yang, Jing Yao, et al. Optogenetic control of RNA function and metabolism using engineered light-switchable RNA-binding proteins[J]. Nature Biotechnology, 2022, 40(5): 779-786.