Multichromatic control of gene expression in Escherichia coli

Abstract

Light is a powerful tool for manipulating living cells because it can be applied with high resolution across space and over time. We previously constructed a red light-sensitive Escherichia coli transcription system based on a chimera between the red/far-red switchable cyanobacterial phytochrome Cph1 and the E. coli EnvZ/OmpR two-component signaling pathway. Here, we report the development of a green light-inducible transcription system in E. coli based on a recently discovered green/red photoswitchable two-component system from cyanobacteria. We demonstrate that the transcriptional output is proportional to the intensity of green light applied and that the green sensor is orthogonal to the red sensor at intensities of 532-nm light less than 0.01 W/m(2). Expression of both sensors in a single cell allows two-color optical control of transcription both in batch culture and in patterns across a lawn of engineered cells. Because each sensor functions as a photoreversible switch, this system should allow the spatial and temporal control of the expression of multiple genes through different combinations of light wavelengths. This feature aids precision single-cell and population-level studies in systems and synthetic biology.

Figure 1

Engineered two-color light induction system in E. coli. (A) Schematic representation of the system. The green sensor and chromophore biosynthetic pathway are as described in the main text. The red light sensing protein Cph8 is expressed from the P_LTetO-1 promoter in the phosphorylated ground state. It is switched to the unphosphorylated state by 650nm, and back to the phosphorylated state by 705nm light . When phosphorylated, Cph8 passes a phosphoryl group to OmpR which then binds to and activates transcription from P_ompC. Because it is inactivated by red light, Cph8 can be considered a logical (NOT red) sensor. A genetic inverter, or logical NOT gate is used to invert the response of the (NOT red) sensor to that of a red light sensor. (B) Plasmid maps of the green + red sensor plasmid pJT122, the red light inverter plasmid pJT106b and pPLPCB(S), a variant of pPLPCB in which the kanamycin resistance cassette has been replaced by a spectinomycin resistance cassette (Materials and Methods). Note that the true configuration of the DNA encoding this system is represented by the plasmid maps while the version shown atop this Figure is slightly simplified for clarity.

Figure 2

Transcriptional response of green and red sensors to different light conditions. (A) E. coli cultures were grown in the dark, under 0.080W/m² 532nm light or 0.080W/m² 650nm light. (+PCB) strain JT2 carrying the green (pJT118) or red sensor (pCph8 + pJT106b3) plasmids and pPLPCB(S). (−PCB) JT2 carrying only the green or red sensor plasmids. Each data point represents the average of four separate cultures grown and measured in parallel on a single day. Data taken under different light conditions were collected on different days. Miller Assays are conducted as reported previously . Error bars represent ± one standard deviation. (B) Plate-based assays of green and red sensors. The mask shown was used to project an image of 532nm or 650nm filtered light onto an agarose embedded film of bacteria expressing the green (top) or red (bottom) sensors. The chromogenic substrate S-gal (Sigma) and ferric ammonium citrate are added to the agarose media such that the product of lacZ, ß-galactosidase, produces a visible black pigment when expressed. 0.030 W/m² 532nm and 0.080W/m² 650nm red light were projected through the mask for all trials. A slightly lower 532nm intensity was used because the red sensor shows a minor response to 0.080W/m2 532nm light (Figure 2a, 3a). The green sensor strain is the same as Figure 2a. The red sensor strain is JT2 carrying pCph8, pPLPCB(S) and pJT106b (a variant of pJT106b3 with a stronger ribosome binding site upstream of lacZ) for higher pigment production on plates. The −PCB condition indicates a given strain lacking pPLPCB(S) exposed to its cognate light wavelength. After 21 hours, the bacterial plates produce images that can easily be seen by eye with no further image enhancement. (C) Spectral transfer functions. E. coli carrying the green or red sensor (strains as in Figure 2a) were exposed to saturating levels of a given light wavelength and Miller Assays were conducted as in Materials and Methods. Data are reported as fold induction over dark exposed cells. This is calculated by dividing the Miller Unit (M.U.) value of the light exposed cells by the M.U. value of the same strain grown in the dark. Each data point represents the average of four separate cultures grown and measured in parallel on a single day. Data at different light wavelengths (or dark) were collected on different days. Error bars represent ± one standard deviation. Miller Assays are conducted as reported previously .

Figure 3

Two-color optical control of gene expression in E. coli. (A) Light intensity transfer functions of strains carrying each sensor alone or both sensors. Strains expressing the green sensor only (green circles), red sensor only (red squares) or both (grey circles) were exposed to varying intensities of 532nm or 650nm light and Miller Assays were conducted as in Materials and Methods. The green and green + red data (circles) obey the left axis while the red sensor data (squares) obey the right axis. Two axes were used because the absolute Miller Unit output of the RBS weakened red sensor is low compared to the green sensor. Error bars represent ± one standard deviation. (B) Two color bacterial photography. A two-color mask was generated from a color-enhanced photograph of chili peppers. Green light passing through the stem regions of the image was set at 0.02 W/m², slightly above the saturation point of the green sensor. At these illumination levels the mask transmits 0.02-0.025 W/m² 650nm light, above the saturation point of the red sensor. The same light intensities were used for all three plates. Green and red sensor only strains are as in Figure 2a. Green + red strain is JT2 carrying plasmids pJT122, pJT106b3 and pPLPCB(S).