pBAD-dCas9-J23119-M+

This composite part is the principal design of the inducible CRISPR-based DNA replication interference system, with the 20 bp sgRNA targeting to the M+(top strand) DnaA box on E.coli genome replication initiation region, OriC. In natural situations, M+ is a low affinity box for DnaA binding. By blocking the binding of DnaA protein to M+ box, moderate arrest and inhibition of genome replication initiation is achieved. For more detailed information, see BBa_K3081058.

Design

Based on CRISPR-interference method for transcription inhibition, we develop a novel approach for prokaryotic genome replication interference (CRISPRri). Hence, a 20-bp sgRNA is designed to be complementary to OriC, the genome replication origin (Figure 1). Instead of site-directed mutations one by one, CRISPRri allows for 20-bp scan each time. Although CRISPRri requires a PAM ("NGG") sequence to execute its function, we found a high occurrence frequency of PAM in the region of replication origin and all available sgRNAs can cover 76.2% (221 out of 290) of OriC.Seven different targeting sites for dCas9 is designed to test the effect on cell growth. We characterized M+ box in detail.

Figure1. Designed targeted box of CRISPRri and observation methods. Functional DNA boxes located on the genome replication origin. The diagram includes high-affinity DnaA binding boxes (R1, R2 and R4), IHF binding site and region for DNA unwinding. Low-DnaA-binding-affinity boxes, R3 and M, are not shown here. Among these boxes, utilized in the experiment are R1, R3, M, IHF binding box and a target box located at the unwinding site (MR13). Another target box, which is located at the linker sequence between M and R2, is also designed. Control group is poly-adenine.

Properties

To precisely record the bacteria growth under stable conditions, a microfluidic chip is developed to adapt to observed features of bacteria (Figure 2). All repeat groups are under flow of the same culture to ensure that the experiment results will not be affected by irrelevant external conditions. We have pointed out that interference of genome replication initiation would result in longer cell cycle and cell number doubling time. Here we take a 90 um * 90 um microscopic view each repeat group for cell counting every half an hour. It turns out that CRISPRri targeted to different boxes on OriC results in variant levels of cell doubling time extension, even though intervals between these boxes are only tens of base pairs. This is consistent with our expectations based on literatures, that functions and essence of different DNA boxes on the OriC and their contributions to genome replication vary a lot. Combined with known mechanism in DNA replication initiation, it is found out that our results accord with the DnaA binding affinity reported previously. High DnaA affinity boxes, like R1 and R3, were shown to have severe inhibition effect when targeted by dCas9. For typical low affinity box, like M box, the effect of CRISPRri is much milder. The only exception is R4, which was reported to be a high-affinity box but shows slight effect on cell growth.

Figure2. Microscopic GIFs of bacteria transformed with CRISPRri system targeted to M+ boxes from microfluidic system. Transformed Top 10 strain is transferred to M9 medium in the ratio of 1:10 after overnight cultivation in LB medium. About 2 hours after transferring, bacteria in its log phase is precipitated by 5000-rpm centrifuging for 4 min and is re-suspended by M9 medium arabinose. Re-suspended bacteria are injected into the chip and observed and recorded continuously for 10 hours, under constant flow of 1 mL/16 hours.

Development, Characterization and Optimization of CRISPR-Based DNA Replication Interference (CRISPRri)

we finely tuned the CRISPRri on multiple aspects , including plasmid copy number, inducer, targeted boxes and other extension for wider and smarter use of the system. Cell number doubling time, nucleo-cytoplasmic ratio, morphology and irrelated protein productivity are seen as the outputs of the system and are all well described and tuned.

We picked up M+ box as the target site of dCas9 due to its relatively milder effect on cell growth, which avoids over-inhibition under low inducer concentration. Another important reason for choosing M+ box instead of other mild boxes like IHF box, is that its relation with DnaA binding shows a clear picture on how DNA replication initiation is delayed and might have higher predictability. For a same reason, we chose the vector with medium copy number to carry the dCas9 gene and sgRNA. We found an arabinose-dose-dependent increase in cell number doubling time with a considerable dynamic range (Figure 3). This realizes preliminary adjustment of bacteria division time.

Figure3. Cell number change of bacteria with CRISPRri targeted to M+ box under induction of different arabinose concentration. Cell counting is finished in hemocytometer every 2 hours.

Measuring the 600-nm light absorbance is a traditional approach for rapid and real-time measurement of bacterial concentration. We found no remarkable decrease in OD600 for bacteria solution treated with CRISPRri (Figure 4A), which is attributed to decrease in cell number but lengthening in cell morphology. As a proof of this concept, we calculated the total covering area of bacteria in microfluidic system, and found the same results (Figure 4B). CRISPRri-implanted E. coli is able to reach a length of 100μm on average and over 500μm at most.

CRISPRri Enhances Cell Adhesiveness to Surface Through a Harmless Lengthening in Morphology

One of the biggest advantages for long-shape-type cell is that its membrane area per cell increases. This allows a stronger interaction between a cell and other interface, like surface of human intestinal tract or genital tract. Longer bacteria might be more stable and easy-to-plant compared to normal-sized cell. As a simplified model, we test the adhesiveness of E. coli with a uniquely designed microfluidic chip (Figure 5A). Flow velocity is adjustable by the injection pump. Long cells and normal cells are mixed in the same culture and are placed into the chip. We found ratio of long cells to normal cells greatly increases as the flow gets faster (Figure 5B).

Irrelative Protein Productivity can be Largely Enhanced in CRISPRri System

As we have mentioned before, CRISPRri system inhibits the cell growth but not in the way of reducing its total biomass. This typical feature means its potential industrial application will not be limited. Furthermore, since cell energy consumption in DNA replication is reduced but nutrition uptake rate might be a constant, production of proteins that has no direct relation with DNA replication would increase. This was proved by measuring the production of GFP per cell mass, which is calculated by GFP fluorescence divided by OD600. We found more than three-fold increase in GFP productivity, and a dose-dependent phenomenon suggests it is caused by CRISPRri system (Figure 6).

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 7). 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.

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 8). Receiver colonies located nearer to the donor grow much slower than farther ones, which is observed in either dropping or smearing plate.

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.

Sequence and Features

Assembly Compatibility: