Light-mediated regulation of gene expression has become a rapidly advancing area of research across many fields of science and engineering. In contrast to chemical inducers, light provides switchable gene expression without a change in metabolic conditions and is an easy-to-use, inexpensive inducer. Here, we developed a novel light-switchable gene expression system that uses the λ cI protein as a transcriptional repressor of target genes.
We also found that the most important parameters that determine the extent and duration of the light-switchable gene expression. The light-switchable promoter systems could be used effectively in metabolic engineering and synthetic biology.
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Nomenclature
Introduction
The araBAD promoter (PBAD) system is a more tightly regulated gene expression system than the lac promoter system. Recently, a propionate-inducible promoter system (PprpB) has been developed for tightly regulated gene expression using a non-toxic and inexpensive inducer, propionate17. Since most chemical inducer-based gene expression systems are derived from E. coli promoters, metabolic load and noise due to cross-talk with the host system is another potential problem.
Distinctive physical properties of light provide a high level of flexibility to control gene expression without disturbing the normal metabolic activities of a cell20. This system was used to demonstrate light-regulated gene expression in E. coli using lacZ as a reporter gene. Analogous to this, the different wavelengths, intensities and duration of light that affect gene expression must be determined.
This system would therefore favor the control of gene expression from multiple promoters with a CI operator site. Our results show that gene expression even from the chromosomal DNA can be controlled by light. Since the duration of photoconversion is very short (usually on the order of picoseconds)26, synthetic biology exploits the rapid response of light to control gene expression.
Several chimeric photoswitches have been developed for differential control of gene expression with a specific wavelength of light such as red, green and blue light27-29. The phosphorylated response regulator in the cytoplasm can then bind to its operator sites to activate or repress gene expression. For example, the acyl homoserine lactone (AHL) inducible promoter system can help modulate gene expression efficiently in a chemostat41.
Materials and Methods
For cI integration, the kanamycin cassette and the cI gene were fused using SOE-PCR61. The cells were chilled immediately after induction to arrest the metabolic state of the cell, made electrocompetent and transformed with the PCR products described above. SOC medium was added after electroporation and the cells were recovered from electric shock by incubation at 30°C for 1 h 30 min.
The kanamycin cassette was then removed using the Flp recombinase system carried on the plasmid pCP2061. The Flp recombinase excises the kanamycin gene flanked between the FRT sites from the chromosomal DNA. PompC-driven LVA-tagged cI gene was integrated into the chromosome of wild-type MG1655 and the chromosomal envZ was deleted to link the expression of cI through the native envZ-ompR two-component signal transduction system.
P1 and P4 of the FRT (FLP recombinase recognition target) sites surrounding the kanamycin resistance gene in pKD13 are linked to H1 and H2 which are the specific homologous region upstream and downstream of envZ. λ-red recombination proteins catalyze recombination as a result of partial envZ replacement with H1-P1-KanR-P4-H2. The last step is to remove the kanamycin resistance gene through the Flp recombinase system in pCP20.
Two PCR products are initially constructed; the first contains cI-LVA flanked by H1 and P4, and the second contains the removable kanamycin resistance cassette flanked by P4 and H2. The activity of the enzyme, β-galactosidase, encoded by the reporter gene lacZ, can be assayed using the substrate, o-nitrophenyl-β-D-galactoside (ONPG) as described previously63. 50 μL of the overnight cultured cells were inoculated into 5 mL of fresh LB medium in culture tubes either wrapped (dark) with foil or unwrapped (light).
Results
We analyzed the effect of light on holo-Cph8 by measuring the LacZ activity in strain RU1012 ( Figure 8 ). We used a regular LED desk lamp (LS-100) to activate the light-sensing holo-Cph8. To investigate the optimal combination of promoter and RBS for tight regulation of a target gene expression, we constructed 4 different reporter plasmids with different promoters/RBSs: PL(RBS0.3), PCP6(RBS0.1), PCP12(RBS0. 1) and PL(RBS0.1) (Figure 10).
In order to determine the relationship between the light dose and the induction fold, GFP expression (under the control of various promoter/RBS combinations) was measured in relation to changes in light exposure duration. The light exposure time was varied sequentially from 0 h to 12 h in strain JM1012 using the LabVIEW instrument. CI-LVA repressor efficiently controlled GFP expression from the weak promoter PL-RBS0.3 in response to diverse light conditions (Figure 12a and 12e).
These results suggest that the high background expression observed in the light-switchable promoter system may be related to the strength of the promoter or RBS. PCP12-RBS0.1 was functional and comparable to PCP6 when tested independently of the CI repressor (Figure 10). The blue line represents the constant presence of light; The red, green, and purple lines indicate 1, 2, and 3 hours of darkness, respectively.
Blue line represents the continuous presence of light; Red, green and purple lines indicate one, two and three hours of light respectively. CI-independent GFP expression from the plasmids (PL-RBS0.3-GFP-LVA, PCP6-RBS0.1-GFP-AAV, and PCP12-RBS0.1-GFP-AAV) appears to be detectable only for 4 to 6 hours . the late log phase (Figure 14). This result indicates that the LVA tag is degraded faster than the AAV tag (PCP6-RBS0.1-GFP-AAV compared to PCP6-RBS0.1-GFP-LVA).
Discussion
Our results indicate that the sensitivity of holo-Cph8 appears to be independent of the wavelength of light. Therefore, this broad sensitivity of holo-Cph8 could be an industrially important property, as there is no need to precisely control the light spectrum. Oscillation of gene expression induced by chemical inducers is usually a discontinuous process where the inducer must be washed away or the cell must be diluted.
We have demonstrated efficient oscillation in gene expression in a continuous process using light-switchable gene expression. To the best of our knowledge, only a switchable promoter system could favor fluctuations in gene expression in a continuous process. This result indicates that gene expression even from chromosomal DNA can be controlled by light.
Similar to a chemically inducible promoter, where expression depends on the concentration of the inducer, the light-switchable promoter system is controlled by the time of light exposure. It is necessary to understand how the combination and abundance of the promoter and RBS affect gene expression for more precise control of multiple genes in synthetic networks72. As well demonstrated in this study, light can provide a temporal change in gene expression.
Use of light-switchable gene expression system may favor a temporary change in gene expression and thus may favor inactivation of these genes only during the stationary phase. Acetate production is thought to be one of the main causes of impaired heterologous gene expression. For example, here we demonstrate the efficiency of the global repressor CI to regulate multiple genes.
Conclusion
It is also not clear whether CCR is imposed at the transcriptional level or at the translational level79. Similarly, Catabolite Repressor Protein (CRP), the global regulator of CCR, is known to affect transcription from more than 200 genes in E. If we can induce light-driven CRP expression instead of light-driven CRP expression glucose, then we may be able to maintain a homeostatic CRP level either in the presence or absence of glucose.
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Acknowledgements
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