Experiments

In the lab at Tianjin University, the plasmid containing cI857 gene and its up- and downstream regulatory elements (high-expression promoter PpsbA2 and terminator TrrnB) as well as the cI857-controlled promoter P_R and terminator TrbcL has been already constructed for the temperature-control of another gene previously [3]. This plasmid is named pS1, which also contains the NSIII homologous arm for the homologous recombination of this plasmid into the cyanobacterial genome. Our work is thus to insert the ssr1114 gene into the pS1 plasmid between the P_R promoter and the TrbcL terminator to construct a recombinant plasmid pS1-ssr1114 (figure 3).

Table 1: The conditions and process for fusion-PCR

Table 2: The primers used for fusion-PCR

The antitoxin gene ssr1114 is an endogenous gene of the cyanobacteria strain Synechocystis sp. PCC6803. So we got this gene by amplifying it from the genomic DNA purified from PCC6803. The reaction conditions of the PCR are shown in Table 1 and the primer sequences are shown in Table 2. Since homologous recombination would be used for plasmid construction, the sequence of a pair of homologous arms (the sequence of the P_R promoter and TrbcL terminator) was added to the 3’ end of the pair of primers respectively (the sequence shown in lower-case letters in Table 2).

Then, the purified ssr1114 gene fragments with the homologous arms were inserted into the pS1 plasmid by homologous recombination with the help of the reagent 2×One Step Fusion Cloning Mix (TOROIVD®). The reaction system is shown in Table 3. After incubating the reaction mixture system at 50℃ for 30 min, the mixture solution was transformed into E. coli competent cells. When colonies appeared on the culture medium, colony PCR was used to test whether the target gene had been successfully integrated into the plasmid. The electrophoresis result of the colony PCR is shown in Figure 5. Compared to the marker, the length of the PCR product is line with our expectation. The following DNA sanger sequencing (Azenda life sciences, Ltd. Co.) for the extracted plasmid further verified the successful construction of the recombinant plasmid pS1-ssr1114 (Figure 3).

Table 3: The reaction system of homologous recombination cloning

Figure 6. The electrophoresis results for the amplified ssr1114 gene inserted in pS1 (middle) and the amplified slr0664 gene inserted in pS2 (right); The left bands are DNA standard ladders (Thermo ScientificTM GeneRuler 1kb Plus DNA ladder).

The gene slr0664 was also amplified from the genomic DNA from Synechocystis sp. PCC 6803 with the primers shown in Table 2 (the sequence shown in lower-case letters are the homologous arms for recombination cloning). The procedure for the construction of pS2-slr0664 plasmid (Figure 4) was the same as the construction of the pS1-ssr1114 plasmid (Table 1), but the primers for ssr1114 were replaced by the primers for slr0664 (Table 2) and the plasmid vector was replaced by pS2. The integration of the target gene was also tested by colony PCR after transformation. The result of electrophoresis is shown in figure 6. Then, the following DNA sequencing indicated a successful construction of the pS2-slr0664 plasmid (Figure 4).

Figure 7. Team members were doing experiments for the construction of plasmids

The pS1-ssr1114 and pS2-slr0664 plasmids were then introduced into wild-type cyanobacteria strain Synechocystis elongatus PCC 7942 by transformation with the following procedure:

(1)Transform the two plasmids into two tubes of trans 5α E. coli competent cells respectively;

(2)Cultivate the wild-type S. elongatus PCC 7942 cells in BG11 liquid medium for 2 days till the OD750 reach to 0.5;

(3)At 4-5 hours before transformation, activate the donor trans 5α E. coli (transformed with pS1-ssr1114 or pS2-slr0664) and Helper (HB101with assistant plasmid pRL443 and pRL623) and inoculate them with the ratio of 1:50 and 1:100, respectively, into 5ml LB liquid medium until the OD630 reach 0.3;

(4)The donor E. coli and Helper were then centrifuged at 6,500rpm in a refrigerated high-speed centrifugator at 4℃, and then washed with LB liquid medium for 3 times in order to remove the antibiotics in the medium;

(5)Incubate the washed donor E. coli and Helper at 37℃ for 30min.;

(6)The wild-type S. elongatus PCC 7942 cells were also washed with BG11 liquid medium in the same way so as to remove the antibiotics inside;

(7)The washed cyanobacteria were resuspended with 200 mL BG11 liquid medium and added to the incubated culture medium with donor E. coli and Helper, then the whole mixture was incubated for 30min. at 37℃;

(8)The medium with the mixture of 3 different bacteria was then smeared evenly on the solid medium without antibiotics (95% BG11 and 5% LB) covered with cellulose acetate membrane and cultured in illuminating incubator at 37℃ for 1 day;

(9)Move the cellulose acetate membrane into the BG11 solid medium with antibiotics and cultivated in illuminating incubator at 37℃ for 4-5 days until colonies emerged on the medium;

(10)Colony-PCR was then applied to identify colonies containing our target genes;

Figure 8. Team members conducted the plasmids transformation into the wild-type S. elongatus PCC 7942

For the construction of the plasmid pANL-trc-DR-crpilO-psbA2- Asddcpf1-kan to arrest cyanobacterial mobility, we designed the crRNA sequence agianst pilO gene, which is shown in Table 4. To insert the gene behind the DR sequence, we used the type II restriction enzyme Esp3I, whose cutting site is depicted in Figure 9, and performed the ligation using T4 DNA ligase. The product of ligation was then purified and the successful insertion of the target gene was confirmed by PCR amplification against the target gene. The electrophoresis result is shown in figure 10.

Figure 9. The cutting site of the restriction enzyme Esp3I.

Figure 10. Electrophoresis of the amplified crRNA gene inserted in pANL-trc-DR-crpilO-psbA2- Asddcpf1-kan.

Table 4. The sequence of the gene encoding the crRNA against pilO gene

The newly-constructed plasmid was transformed into cyanobacterial cells (the method has been described above). To test the effectiveness of constructed plasmid in cells, the selected homologous cyanobacteria strains’ mobility was monitored and recorded under optical microscope following the procedures described below:

Step one: Preparation of Ibidi Microfluidic Chips

In order to observe cyanobacterial movement under microscope, an Ibidi microfluidic chip device is used. So the first step is to assemble and clean the chip device.

(1) Clean the channels of an Ibidi microfluidic chip and a cover glass with ultrapure water and anhydrous ethanol, then dry them with nitrogen;

(2) Use a 10 mL syringe to draw an appropriate amount of glass glue (Dow Corning 732), cut the tip of a 20 μL syringe nozzle at an angle, remove the rear part, and attach it to a 1 mL syringe nozzle, then load the glass glue into the 1 mL syringe to ensure even application;

(3) Using the glue-loaded 1mL syringe, apply a uniform layer of glue around the edges of the Ibidi microfluidic channels; quickly place the cover glass on the surface of the channels; then invert the device onto a smooth surface and press the Ibidi chip gently and uniformly to ensure that the cover glass is attached to the Ibidi chip firmly and the Ibidi channel is sealed by the glue and cover glass;

(4) Let the device stand still for 12 hours to allow the glass glue to dry completely;

(5) Prepare two pieces of tubing with an inner diameter of 0.8 mm (approximately one 30cm-long, and the other 40cm-long), a 0.22 μm micropore filter membrane, a 50 mL syringe, and connectors;

(6) Connect parts sequentially in the order of syringe, micropore filter membrane, tubing, connector, Ibidi microfluidic channel inlet, connector, tubing, and waste collector; Note that the waste collector and the Ibidi microfluidic channel are at the same level to prevent backflow. Figure 16 shows an example of an assembled device.

(7) The assembled microfluidic device need to be sterilized. Fill the syringe with 3% hydrogen peroxide, assemble it with the rest of the system as described above, and use a syringe pump to push the syringe to wash the assembled system at a flow rate of 3 mL/h for 6 hours.

(8) After sterilization, replace the syringe with one filled with sterilized deionized water and change a new micropore filter membrane, then further flush the system at a flow rate of 3mL/h for 6 hours to wash away hydrogen peroxide.

(9) After sterilization and cleaning, the device is ready to use. Replace the syringe with one filled with the required culture medium and change a new micropore filter membrane to prepare for the next step of aimed experiments.

Figure 11. An assembled microfluidic device consisting of syringe, filter, Ibidi chip, and waste collector.

Step two: Microscope Data Collection

After the microfluidic chip device is ready to use, we then perform the following procedures to collect the microscope data for cyanobacterial movement.

(1) Cultivate cyanobacteria in a photo incubator to the logarithmic phase, with an OD750 of approximately 0.4, then draw 1 mL culture for later usage;

(2) Collect data using a Leica microscope with a CCD camera (Andor CCD NEO), integrated with Leica software. Turn on the microscope and camera in sequence, set the temperature of sample stage to 37°C, and open the microscope software;

(3) Install the syringe of the Ibidi microfluidic device on a syringe pump, apply a drop of immersion oil vertically on the 100× oil objective, place the Ibidi microfluidic channel on the objective, and secure it with the stage apparatus. The experimental set-up is shown in Figure 17.

(4) Adjust the coarse focus knob to bring the objective lens close to the cover glass of the Ibidi microfluidic channel, then adjust the fine focus knob;

(5) Before inoculating bacteria, take a single picture as a background image for later background correction;

(6) Using a 1 mL syringe, inject 300 μL of diluted culture (OD=0.4) into the Ibidi microfluidic channel through the connected tubing (stop the flow during injection, and clamp the inlet tubing with a clip to prevent backflow of the culture toward the syringe);

(7) After inoculation, let the device stand still for 10 minutes to allow cyanobacteria to settle down to the glass surface; then set the flow rate to be 20 mL/h to flush away unattached cyanobacteria; after 5 minutes, stop the flow.

(8) Adjust the fine focus knob so that bacteria in the field of view can be observed clearly; select a field of view with an appropriate number of bacteria; hold the focal plane to ensure it does not drift; set the recording parameters, such as the recording rate of 1 frame per second (fps), and the total recording time of 20 minutes.

(9) Start recording. The recoding will be finished once the total recording time reaches the set value.

Figure 12. Experimental setup diagram for collecting cyanobacterial movement data.