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[1]. 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+ box 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.

Figure4. Biomass of CRISPRri-implanted bacteria (A) Time-course OD600 of bacteria with CRISPRri targeted to M+ box under different arabinose concentration, tested by microplate reader. (B) Normalized covering area of M+ group bacteria in a microfluidic chip chamber, under flow of M9 medium with different arabinose concentrations. Covering area at the beginning is treated as 1. 8 chambers is measured for each group.

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).

Figure5. Measurement of cell adhesiveness. (A) A uniquely designed microfluidic chip as a platform to measure cell adhesiveness. The preparation workflow is as follows: (a) Use a puncher to make two holes at proper locations, according to the structural parameters shown here. (b) Stick a double-sided adhesive tape to fully cover the area of cover glass and the tape covering the view field is removed (the dotted box). (c) Add the cover glass onto the glass slide. (d) Fasten the slide with two clamps and heat the slide in 200 degree Celsius in vacuum. (e) The chip is available after natural cooling. (B) A simple experiment is designed to test the cell adhesiveness. E. coli transformed with CRISPRri system targeted to M+ box and induced by 0.50% arabinose M9 medium. Then the elongated M+ group cells is mixed with normal-sized PolyA group to obtain a long and short cell mixture. The mixed bacterial solution is injected into the chip and stand for 20 min to ensure the sedimentation of most cells. After that, the injection pump constantly provides the liquid buffer with a settled flow velocity. The initial velocity is 1 mL/4 hrs, and is slowly developed by 2-fold. Microscope would record the process until the flow velocity reaches the maximum of the injection pump. We defined a cell to be long if it takes more than 80 pixels in a microscopic graph. We counted the ratio of long cells to normal cells every time we develop the flow velocity. Initial ratio is normalized to 1.

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).

Figure6. GFP production per cell mass unit. CRISPRri system targeted to M+ is co-transformed with a constantly-expressed GFP plasmid. Fluorescent intensity and OD600 are both measured by microplate reader. The transformed strain is transferred to M9 medium after overnight cultivation. Three hours after transferring, the bacterial solution is diluted into medium with different arabinose concentration. The ratio of FI to OD600 shown here is at the timepoint of 6 hours.

Figure7. Indigo production of E. coli with or without CRISPRri.(A) Schematic diagram of indigo synthesis process. (B) Photo of indigo production in M+ group(left) and polyA group(right). (C) Quantitative results of samples shown in Figure 7B.

The CRISPRri system can work well to control the growth of E. coli Nissle 1917 strain.

Escherichia coli strain Nissle 1917 (EcN) is a remarkable probiotic bacterium, which has been well researched over decades. Considering the non-pathogenicity of Nissle strain, it is widely used as a chassis organism in current industrial applications and biological therapies. Considering that the oriC sequence of E. coli Nissle is identical to that in Top10, we hope to demonstrate if the function of CRISPRri system in the new E. coli strain is same as it in Top10.

We transformed CRISPRri system to E. coli Nissle 1917. Through the microfluidic imaging system, we observed that with the treatment of 0.25% arabinose, the proliferation of bacteria in the experimental group, whose sgRNA targets on the M box in oriC region, was significantly slower than control groups. Quantitative results showed that the average cell number doubling time of the experimental group (M+) was greater than 2 hours, while the control group (polyA) only need about 40 minutes to complete cell division.

As mentioned before, irrelative protein productivity can be largely enhanced in bacteira with CRISPRri system. We co-transform GFP-expression plasmid and CRISPRri system into one cell. We found the fluorescent expression intensity of bacteria in the experimental group (M+) increased significantly after treatment with arabinose inducer compared with no arabinose treatment, but the control group did not have such a phenomenon (Figure 8 GIF).

T--Peking--nissle-M%2Bgif.gif M+ PolyA

Figure8.Characterization of CRISPRri system in Nissle, with dCas9 targeted to M+ or polyA boxes.

（Considering the technical problem, if the reader is interested in our results, welcome to our presentation and poster session to watch the original movie.）

Reference

[1]Wolański M, Donczew R, Zawilakpawlik A, et al. oriC-encoded instructions for the initiation of bacterial chromosome replication.[J]. Frontiers in Microbiology, 2015, 5(735):735.

Sequence and Features

Assembly Compatibility: