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Revision as of 14:35, 28 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 deassiociate and lose the ability to repress gene expression. Therefore, the downstream genes will be expressed.

Usage and Biology

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.

Characterisation from HKUST-GZ 2024

Characterisation

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.

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.

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.

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.

References

• 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. [2]
• 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. [3]
• Camsund, D., Jaramillo, A., & Lindblad, P. (2021). Engineering of a Promoter Repressed by a Light-Regulated Transcription Factor in Escherichia coli. BioDesign Research, 2021. [4]
• 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. [5]
• 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. [6]

Sequence and Features