Part:BBa_K3081054

Quorum Sensing CRISPRri System Enables Spatial-level Growth Control and Ultrasensitive Autoregulation of Growth

Hence, based on the well-characterized growth control system, we constructed a quorum sensing CRISPRri system (qs-CRISPRri) to realize smart regulation of bacterial overall states.

A time-scale quorum sensing system can be transformed to spatial-level through a donor/receiver system (Figure 1). The donor cells, which merely express and release AHL, would activate the GFP expression of receiver cells through AHL diffusion. This is validated on the agar plate, by dropping the donor cells in the center and receiver cells around them with different distances. We found a progressive decrease in fluorescence as the receiver cells locate farther from central AHL donor.

Figure 1.The donor-receiver split quorum sensing system of CRISPRri system. Upper figure is the gene circuit of donor and receiver cells of qs-CRISPRri system. For the figure below, a donor-receiver split quorum sensing GFP system enables fluorescent intensity control on spatial scale. Left figure is the sketch map of how donor and receiver bacteria is dropped onto the agar plate. Right figure is the real picture of agar plate under illumination of blue light. The location of the white arrow is the donor cells. Receiver cells are marked by number one to six.

GFP gene is exchanged for dCas9 with companion of constantly-expressed single guide RNA to establish the qs-CRISPRri system. Through the donor-receiver system, we realized spatial-level control of cell growth (Figure 2). Receiver colonies located nearer to the donor grow much slower than farther ones, which is observed in either dropping or smearing plate.

Figure 2.Spatial-level growth control of donor-receiver split qs-CRISPRri system. For both two agar plates, the colony located at the center is the donor cells.

Furthermore, a complete quorum sensing system-implanted strain is tested with microfluidic system. Distinct from arabinose-induced CRISPRri system, decrease in cell division rate shows an ultrasensitive behavior. The cells were found to almost stop division and only lengthen its own shape all of a sudden, which we believe to be attributed to accumulation of AHL. Since cell growth is exponential at early stage, AHL concentration would increase rapidly. As AHL concentration reaches a certain threshold, cell genome replication is interrupted by binding of dCas9 and cell cycle extends largely. This enables E. coli to rapidly autoregulates its own growth state, and provides a potential concept of self-tuning medical-used bacteria without external interference.

We hope to construct a self-regulating system, but in our experiment, the expression of LuxI is still artificially induced by IPTG. The only reason why we do not use a constant promoter is that we try to avoid over-inhibition of cell growth during molecular cloning. In fact, it can be replaced by any constant promoters with proper strength to realize full-automatic regulation of growth in real application.

Reference:

[1]Weiqian Zeng, et al. Rational Design of an Ultrasensitive Quorum-Sensing Switch. ACS Synthetic Biology 2017 6 (8), 1445-1452.

[2]Y. Soma, T. Hanai. Self-induced metabolic state switching by a tunable cell density sensor for microbial isopropanol production. Metabolic Engineering 30 (2015) 7–158.