Difference between revisions of "Part:BBa K5280129"

Revision as of 13:28, 29 September 2024

LexRO

LexRO is a recombinant protein which will dimerise in darkness and the dimers will bind to DNA, blocking the expression of downstream genes. When exposed to light, more specifically blue light (450~465nm), the dimers will dissociate and lose the ability to repress gene expression. Therefore, the downstream genes will be expressed.

The corresponding regulatory promoter is BBa_K5280401. However, actually all promoters which can be regulated by LexA(408) can be used as the downstream promoter of LexRO.

Sequence and Features

Usage and Biology

This regulator, LexRO, was firstly published in the article on Nucleic acids research。

LexRO is a recombinant protein consisting of 3 parts: a DNA-binding domain from LexA(408), a light sensing domain from RsLOV, and the linker between them.

• RsLOV is a blue light (450~465nm) sensor from Rhodobacter sphaeroides that possesses a contrary light-inducible behavior to Vivid domain. [1]
• LexA(408) repressor is a mutant of LexA that recognizes a symmetrically altered operator mutant but not a wild-type operator. Thus, as long as a promoter can be repressed by LexA(408), it can be regulated by LexRO.

Usually we use PcolE408 (see BBa_K5280401 for more information) as its corresponding downstream promoter.

It is a highly tunable light sensor whose threshold and sensitive wavelength can be modified by tuning the expression level of the regulator.

This image shows the working mechanism of LexRO and its components.

Characterisation from HKUST-GZ 2024

Group: HKUST-GZ

Author: Hua XU

Characterisation

This year we HKUST-GZ iGEM team characterised its cell toxicity, switch ratio and time-course characteristics.

Cell Toxicity

Conditional control of gene expression through regulatory elements requires initial consideration of whether the protein expression will affect the normal physiological functions of the cells. Among the myriad of physiological functions, the ability to grow and develop normally is the most critical. Previous literature has reported that the light-regulatory protein EL222, which shares a similar regulatory mechanism with LexRO, exhibits cytotoxicity in E. coli, inhibiting the growth of biomass in bacterial colonies (Camsund et al., 2021b). To explore whether LexRO possesses cytotoxicity, we designed a series of plasmids and cultured E. coli transformed with these plasmids under suitable conditions, measuring their biomass at fixed intervals. The results are illustrated in the figure below. The findings indicate that strong expression of LexRO does not significantly affect cell division, and significant fluorescence can be observed under cultivation conditions, suggesting that LexRO shares similar fluorescent characteristics with EL222. We note that the excitation wavelength of EGFP is 487 nm, which is very close to the excitation wavelengths of LexRO and EL222, implying that the expression of these regulators might affect the detection of EGFP as a reporter in future applications. Further experiments are still necessary to determine whether there is any impact in real-world scenarios.

In the experiments, E. coli transformed with an empty vector were used as negative controls. Left panel: E. coli transformed with different plasmids were cultivated under optimal conditions, and the OD600 was measured every 20 minutes. Central panel: Fluorescence levels were detected under fluorescence conditions with Ex 485 nm and Em 535 nm, and it was possible to detect fluorescence from LexRO. Right panel: Fluorescence intensity was normalized to OD600, and values from the early stages of cultivation were discarded.

To further ascertain the presence or absence of cytotoxicity and to compare the characteristics with EL222, we cultivated bacteria expressing high levels of EL222 in parallel. The results are illustrated in the figure below. It was observed that there were no significant differences in the growth curves of bacteria expressing an empty vector, EL222, and LexRO, suggesting that LexRO does not exhibit cytotoxicity. It is noteworthy that in our experiments, no significant differences were found in the growth curves between bacteria expressing EL222 and those transformed with an empty vector, which contradicts some references and may be related to variations in cultivation conditions such as light exposure, temperature, nutrition, and antibiotics, warranting further exploration.

Cultivated cells transformed with different plasmids under optimal conditions were synchronized, and samples were taken every 10 minutes to measure the OD600 of the cells.

Effectiveness of Regulation

To assess the efficacy of LexRO as a photosensor, we constructed an expression vector featuring mCherry as a reporter gene. Subsequently, bacteria harboring the reporter gene were cultivated under inducing and non-inducing conditions, with results as illustrated in the figure. Statistical analysis revealed that LexRO, as a regulatory protein for gene expression, can achieve a switch ratio of approximately 6. This performance exceeds that of commonly used optogenetic regulatory elements such as EL222, whose switch ratio is approximately less than 5 folds (Li et al., 2020), suggesting its relatively high efficacy.

Transfected cells with the expression vector were cultured under light/dark conditions, and after 25 hours, flow cytometry was employed to measure the fluorescence intensity and distribution of the cells. The three left panels illustrate the gating methods, while the far-right panel represents the downstream expression intensity using the median fluorescence intensity of the cell population, with three controls for each light and dark group. Using an unpaired t-test, a P-value of 0.0292 was obtained, which is less than 0.05.

Time-course Characterisation

To refine the description of LexRO's regulatory performance on a temporal scale, we characterized the time-course relationship of LexRO under induced and non-induced conditions. The results indicate that during the early stages of growth, LexRO exhibits a high capacity for gene expression repression in the dark, and this repression is relatively complete. For the reversibility group, it was observed that the fluorescence intensity responds significantly to light conditions but with a certain degree of lag. This experiment serves as a preliminary characterization; due to time constraints, we were unable to successfully construct a plasmid for the constant expression of mCherry to serve as a positive control. Additionally, due to limitations in shading conditions, it was inevitable that the sampling process would induce expression in the dark group, which actually interfered with the normal repression process. In this experiment, tin foil was used for shading, but its effectiveness was suboptimal, and damage to the foil occurred. Furthermore, because the blue light lamps used in the experiment generated significant heat, leading to local temperature differences among the experimental groups, the experimental data exhibited noticeable fluctuations in the later stages.

It is important to note that the use of tin foil as a shading material and the challenges associated with it, such as its limited effectiveness and potential for damage, as well as the heat generated by the light source, are factors that can affect experimental outcomes. These factors should be considered and addressed in the experimental design to ensure more accurate and reliable results. Future experiments should aim to improve shading techniques and control for temperature variations to minimize such disturbances. Additionally, the construction of a plasmid for the constant expression of a reporter gene like mCherry would provide a valuable positive control for assessing the regulatory effects of LexRO more comprehensively.

The upper panel data represents the time-course characterization of LexRO, where bacteria transformed with the reporter expression vector were continuously cultured under inducing and non-inducing conditions for 12 hours, with samples taken every 1 hour to characterize their growth metrics. The lower panel data characterizes the reversibility of LexRO regulation. We transferred bacteria with the expression vector and alternated them between inducing and non-inducing conditions at 3-hour intervals, measuring relevant metrics hourly. At the 10th hour of data measurement, a spike in fluorescence intensity was observed in the dark group, suggesting a potential issue with the experimental procedure.

Tunability

Regarding its tunability, researchers have discovered that the sensitivity threshold of a light sensor can be adjusted by manipulating the expression levels of the regulatory protein. This is because the association of two monomers to form a dimer is akin to a reversible reaction, and the equilibrium of such a reaction can be influenced by the concentration of the substrate. By increasing the substrate concentration, we can elevate the level of dimers within the cell, enhance the repression effect, and thereby raise the threshold of the photoreceptor. This approach provides a strategy for fine-tuning the response characteristics of optogenetic tools like LexRO to suit various experimental requirements.

Due to time constraints, we were unable to experimentally validate the tunability of the system. However, we conducted modeling to predict its potential behavior. The figure below presents our predictions. We simulated different promoters with varying expression strengths to drive the expression of LexRO, while maintaining constant light intensity to represent a stable environmental condition. Using our model, we predicted the mRNA levels of the downstream gene. The results indicated that different expression levels of LexRO lead to varying expression levels of the downstream gene, demonstrating that the same regulator can exhibit diverse responsiveness depending on its expression strength.

The upper panel data represents the time-course characterization of LexRO, where bacteria transformed with the reporter expression vector were continuously cultured under inducing and non-inducing conditions for 12 hours, with samples taken every 1 hour to characterize their growth metrics. The lower panel data characterizes the reversibility of LexRO regulation. We transferred bacteria with the expression vector and alternated them between inducing and non-inducing conditions at 3-hour intervals, measuring relevant metrics hourly. At the 10th hour of data measurement, a spike in fluorescence intensity was observed in the dark group, suggesting a potential issue with the experimental procedure.

The contents below are cited from Li and his colleague in 2020.

And the authors of the original paper also did detailed works. As illustrated in the figure below, LexRO exhibits varying responses to light of different wavelengths and intensities, indicating that our photoreceptor has adjustable sensitivity. This feature is crucial for optogenetic applications where precise control over gene expression in response to specific light conditions is required. The ability to modulate the strength of the response allows for fine-tuning the system to match the desired biological or technical outcomes.

The authors modulated the expression levels by utilizing different Shine-Dalgarno (SD) sequences with varying strengths (with SD2 being the strongest and SD37 the weakest in their experiments). They tested the reporter gene expression under various conditions, adjusting both light intensity and wavelength, with the results illustrated in a heat map. The data revealed that as the expression level of the regulator decreases, the repression becomes weaker, resulting in a lower activation threshold for the system.

References

• Li, X., Zhang, C., Xu, X., Miao, J., Yao, J., Liu, R., Zhao, Y., Chen, X., & Yang, Y. (2020). A single-component light sensor system allows highly tunable and direct activation of gene expression in bacterial cells. *Nucleic Acids Research*, *48*(6), e33. [2](https://doi.org/10.1093/nar/gkaa044)

• Jayaraman, P., Devarajan, K., Chua, T. K., Zhang, H., Gunawan, E., & Poh, C. L. (2016). Blue light-mediated transcriptional activation and repression of gene expression in bacteria. *Nucleic Acids Research*, *44*(14), 6994–7005. [3](https://doi.org/10.1093/nar/gkw548)

• Motta-Mena, L. B., Reade, A., Mallory, M. J., Glantz, S., Weiner, O. D., Lynch, K. W., & Gardner, K. H. (2014). An optogenetic gene expression system with rapid activation and deactivation kinetics. *Nature Chemical Biology*, *10*(3), 196–202. [4](https://doi.org/10.1038/nchembio.1430)

• Camsund, D., Jaramillo, A., & Lindblad, P. (2021). Engineering of a Promoter Repressed by a Light-Regulated Transcription Factor in *Escherichia coli*. *BioDesign Research*, *2021*. [5](https://doi.org/10.34133/2021/9857418)

• Dietler, J., Schubert, R., Krafft, T. G., Meiler, S., Kainrath, S., Richter, F., Schweimer, K., Weyand, M., Janovjak, H., & Möglich, A. (2021b). A Light-Oxygen-Voltage receptor integrates light and temperature. *Journal of Molecular Biology*, *433*(15), 167107. [6](https://doi.org/10.1016/j.jmb.2021.167107)

• Ohlendorf, R., Vidavski, R. R., Eldar, A., Moffat, K., & Möglich, A. (2012). From Dusk till Dawn: One-Plasmid Systems for Light-Regulated Gene Expression. *Journal of Molecular Biology*, *416*(4), 534–542. [7](https://doi.org/10.1016/j.jmb.2012.01.001)