Team:XHD-Wuhan-B-China/Proof Of Concept
Light Controlled RNAi
as shown in Fig. 1, the construction of an optically controlled RNAi system is divided into three steps. The first step is to design primers to PCR from the genome of Escherichia coli to obtain dicF fragments. In order to verify that sRNA DicF can indeed inhibit the cell division of MG1655, in the second step, we construct dicF downstream of the arabinose promoter to get pBAD-dicF, and use arabinose to induce the expression of DicF. When it was successfully verified that dicF did inhibit the cell division of MG1655, in the third step, we combine dicF with two-component optical system ccaR/ccaS to get 172_dicF_ccaR&S.
Figure 1. Experimental design flow chart.
In order to realize the real-time observation of 172_dicF_ccaR&S MG1655 strains under the microscope, we have constructed a set of devices. First of all, we use the 3D printer to print the octagonal mold, and then through the design an electronic circuit, the light path and light bulb are installed on the octagonal mold to form our light control device (Fig. 2). We can convert red light to green light through a small switch. Then, we use foam to make a cover that can be placed on the microscope (Fig. 3), which can not only keep warm, but also isolate most of the interference light from the outside world. Finally, two heating devices (Fig. 4) are added to maintain the temperature required by the cell.
Figure 2. Light control device. It can convert green light (left) to red light (right) through a small switch.
Figure 3. A microscope cover made of foam.
Figure 4. Microscopic observation device. Left: monitoring equipment. Right: the microscope adding heater, thermal shield, light control circuit.
Through PCR and recombination, we constructed the pBAD-dicF plasmid (Fig. 5). We transformed pBAD-dicF plasmid into MG1655. In order to ensure that dicF was successfully constructed on pBAD24 plasmid, we designed primers for colony PCR upstream and downstream of dicF. The theoretical size of PCR product is 336bp, as shown in Fig. 6. The gel electrophoresis results showed that the pBAD-dicF plasmid was constructed successfully. After transforming pBAD-dicF into MG1655, when the culture medium OD600 was 0.3, it was induced with arabinose (0.2%) for 12 hours and then observed under microscope (40 ×). As shown in Fig. 7A, compared with the strain pBAD-dicFMG1655 without arabinose (Fig. 7B), most of the strains induced by arabinose were filamentous, indicating that the process of cell division was inhibited. We calculated the relative length of MG1655 cells in terms of the pixels of the microscope photos, and the relative length of cells induced with L-arabinose increased by about 3 times (Fig. 8).
Figure 5. Plasmid map of pBAD-dicF.
Figure 6. Gel electrophoresis of verification of recombinant plasmid pBAD-dicF.
Figure 7. Microscopic (40×) observation map of pBAD-dicF MG1655. (A) Induced by adding L-arabinose. (B) Do not add L-arabinose. (A) and (B) were cultured in 200rpm shaker at 37 ℃ for the same time.
Figure 8. Statistical diagram of MG1655 cell length (In pixels).
After successfully verifying that DicF can indeed inhibit the division of MG1655, we cloned the dicF recombinant into the optical system and obtained the 172_dicF_ccaR&S plasmid (Fig. 9). In order to ensure that dicF was successfully constructed on the optical system plasmid, we designed the primers of colony PCR in the upstream and downstream of dicF. The theoretical size of PCR product is 508bp. The gel electrophoresis results showed that the 172_dicF_ccaR&S plasmid was constructed successfully (Fig. 10).
Figure 9. Plasmid map of 172_dicF_ccaR&S.
Figure 10. Gel electrophoresis of verification of recombinant plasmid 172_dicF_ccaR&S.
The circuit of 172_dicF_ccaR&S is shown in Fig. 11. We hope that when induced by green light of 520nm, DicF is expressed and cell division is inhibited; when induced by red light of 660nm, DicF is not expressed and cells divide normally.
Figure 11. Schematics of the 172_dicF_ccaR&S circuit. Under the green light of 520nm, CcaS (green circle) is self-phosphorylated, then the phosphate group (yellow circle) is transferred to the corresponding regulatory factor CcaR (red circle), and the phosphorylated CcaR binds to the cpcG2 promoter to start the expression of DicF. The process is reversed at 660nm red light.
After the 172_dicF_ccaR&S plasmid was transformed into MG1655, we were ready for real-time microscopic (40×) observation.
Sample preparation：
1. Take the sterilized clean 50mL cone bottle, add 10mL LB and 1.5% low melting point agarose, heating until the agarose is completely dissolved；
2. Take a short-wave plate and put a 24 × 24 cover glass on the short-wave plate, absorb the newly heated agarose LB 1mL and add it to the cover glass, and quickly take another cover glass to cover the agarose (Note: no air bubbles). Wait about 5min until LB solidifies, gently remove the upper cover glass, and cut agarose LB into small square pieces of about 6mm (Fig. 12);
3. The OD600 of 172_dicF_ccaR&S MG1655 cultured overnight was measured and diluted to 0.01;
4. Add 2uL of diluted bacteria solution to agarose mat and dry at room temperature (about 10-15min);
5. Gently transfer the agarose mat to the petri dish, place the side with bacteria upside down against the bottom of the petri dish, and then seal the petri dish (Fig. 13).
Microscopic observation:
1. Install the prepared sample, light control device, heater and thermal shield on the microscope;
2. The growth of MG1655 was observed with a 40× microscope, and the film was set to be taken every 30 seconds.
First, we used green light to induce for about 7.5 hours, obtained 937 photos, and found that the MG1655 had formed a filamentous shape (Fig. 14B). Then, we induced with red light for 10 hours and obtained 1200 photos, at which time some of the cells in the picture had returned to normal division (Fig. 14C). However, the cells in the lower part of the image still appear filamentous, which may be due to the fact that the observed sample is in the center and there are other samples around to block the light. Subsequently, we observed the marginal samples (Fig. 13) and found that almost all the cells in the marginal samples had returned to normal division (Fig. 14D).
In addition, we made two videos from 937 photos induced by green light and 1200 photos induced by red light. Through these two videos, we can directly observe that the cell division stops under the green light induction and resumes the division under the red light induction. The video is at the bottom of this page.
Figure 12. Preparation of agarose mat.
Figure 13. Samples for observation.
Figure 14. Real-time observation microscope (40×) map of 172_dicF_ccaR&S MG1655. (A) Initial cell morphology at 0 h. (B) Cell morphology induced by green light for about 7.5 h. (C) Cell morphology induced by conversion of red light for 12h. (A), (B) and (C) are all intermediate observation objects. (D) Morphology of marginal cells induced by conversion of red light for 12h.