iGEM 2024 — Team thisdl-united

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Notebook

**Laboratory notes for the pre-experiments at Tsinghua International School Daoxiang Lake**

April. 11th, 2024 PCR and DNA purification from gel

![](https://static.igem.wiki/teams/5259/notebook/1.jpg)

![](https://static.igem.wiki/teams/5259/notebook/2.jpg)

April. 18th, 2024 ELISA for enzyme activity analysis

![](https://static.igem.wiki/teams/5259/notebook/3.png)

![](https://static.igem.wiki/teams/5259/notebook/4.png)

May. 23th, 2024 Restriction enzyme digestion and ligation

![](https://static.igem.wiki/teams/5259/notebook/5.png)

**Laboratory notes at Tianjin University**

Construction of the plasmid for toxin protein

![](https://static.igem.wiki/teams/5259/notebook/2024-10-01-11-02-02.png)

![](https://static.igem.wiki/teams/5259/notebook/2024-10-01-11-02-17.png)

![](https://static.igem.wiki/teams/5259/notebook/2024-10-01-11-02-26.png)

![](https://static.igem.wiki/teams/5259/notebook/2024-10-01-11-02-40.png)

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![](https://static.igem.wiki/teams/5259/notebook/2024-10-01-11-02-48.png)

Growth of cyanobacteria

![](https://static.igem.wiki/teams/5259/notebook/2024-10-01-11-13-38.png)

![](https://static.igem.wiki/teams/5259/notebook/2024-10-01-11-13-47.png)

The construction of the plasmid pANL-trc-DR-crpilO-psbA2- Asddcpf1-kan

![](https://static.igem.wiki/teams/5259/notebook/2024-10-01-11-17-40.png)

![](https://static.igem.wiki/teams/5259/notebook/2024-10-01-11-17-46.png)

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Engineering

The toxin-antitoxin system used in the present project is found in the cyanobacteria strain *Synechocystis* sp. PCC 6803 [1]. The toxin gene is named slr0664, which encodes a toxin protein that can attack cell membrane and lead to cell lysis [1]. The antitoxin gene called ssr1114 encodes an antisense RNA to the mRNA of slr0664. The binding of ssr1114 RNA on the slr0664 mRNA results in the degradation of slr0664 mRNA, which stops the synthesis of the toxin [1].

![](https://static.igem.wiki/teams/5259/engineering/1.png)![](https://static.igem.wiki/teams/5259/engineering/2.png)

![](https://static.igem.wiki/teams/5259/engineering/3.png)![](https://static.igem.wiki/teams/5259/engineering/4.png)

![](https://static.igem.wiki/teams/5259/engineering/5.png)![](https://static.igem.wiki/teams/5259/engineering/6.png)

*Figure 1. Our team tried the techniques for cyanobacteria culture at Tsinghua International School Daoxiang Lake (THISDL).*

Therefore, if ssr1114 gene only expresses at or above 37℃, but stops transcription at a temperature below 37℃, we can achieve our goal that the genetically engineered cyanobacteria will suicide in the environment out of the lab when the temperature of the aquatic environment is typically lower than 37℃.

In order to achieve our goal of temperature-controlled transcription of ssr1114, a temperature-sensitive promoter and the gene of its corresponding repressor protein must be assembled together with ssr1114 gene in a plasmid and introduced into cyanobacteria. Through literature search and the instruction of our project advisors from the Synthetic Microbiology Laboratory at Tianjin University, we finally selected the repressor protein cI857 (gene: cI857) and its corresponding promoter PR, which are previously found in bacteriophage lambda (λ) [2]. The repressor cI857 is only activated when the phages (or the host cell) are undergoing a stress of low temperature (lower than 37℃) [2], while a 37℃ temperature in the lab incubator always inactivates cI857 repressor and prevents its binding with PR promoter [2]. Without the binding of cI857, PR is able to bind with RNA polymerase and start transcription of the downstream genes [2]. Thus, if we insert the antitoxin gene ssr1114 behind the PR promoter and then assemble the cI857 gene, which is controlled by a high-expression promoter, we can realize the temperature-controlled expression of ssr1114 (Figure 3).

![](https://static.igem.wiki/teams/5259/engineering/7.png)![](https://static.igem.wiki/teams/5259/engineering/8.png)

![](https://static.igem.wiki/teams/5259/engineering/9.png)![](https://static.igem.wiki/teams/5259/engineering/10.png)

![](https://static.igem.wiki/teams/5259/engineering/11.png)![](https://static.igem.wiki/teams/5259/engineering/12.png)

*Figure 2. Brain-storming of our team under the instruction of the experts of Synthetic Microbiology Laboratory at Tianjin University.*

![](https://static.igem.wiki/teams/5259/engineering/13.png)

*Figure 3. Scheme of the plasmid pS1-ssr1114 (BBa\_K5259000).*

For the toxin gene slr0664, we just needed to express it continuously in cyanobacterial cells. So this gene was inserted between the high-expression promoter PpsbA2 and the terminator TrrnB, which had already been put into a plasmid called pS2 previously. We just needed to insert slr0664 gene behind PpsbA2 promoter. Then, it would be transcribed continuously in cells (Figure 4).

![](https://static.igem.wiki/teams/5259/engineering/14.png)

*Figure 4. Scheme of the plasmid pS2-slr0664 (BBa\_K5259001).*

Given that our results indicated that it took 5 days for the toxin slr0664 to reduce cyanobacteria to meet environmental standards, there remained a risk of the cyanobacteria (or their genetic materials) expanding during this period. To address this, we planned to design a genetic system to restrict the mobility of the cyanobacteria, thereby impeding their expansion within the critical 5-day window before a sufficient number of cells are eliminated. After brainstorming and receiving guidance from our advisors at Tianjin University, we decided to use CRISPR-dCas12a system to knockdown the genes encoding cyanobacteria pili proteins so as to limit their mobility [4]. The plasmid containing PpsbA2 -driven dCas12a gene and the Ptrc1O-driven DR sequence had already been constructed previously at Tianjin University and our task was to design the crRNAs against genes for pili proteins and insert them downstream of the DR sequence (Figure 5). Due to time constraints, to knockdown, the gene encodes PilO, part of inner membrane alignment complex called PilMNOP [5] and to evaluate its effects.

![](https://static.igem.wiki/teams/5259/engineering/15.png)

*Figure 5. Scheme of the plasmid pANL-trc-DR-crpilO-psbA2- Asddcpf1-kan (BBa\_K5259002).*

**References**

[1]Pandey D P, Gerdes K. Toxin-antitoxin loci are highly abundant in free-living but lost from host-associated prokaryotes. Nucleic Acids Research, 2005, 33(3): 966-976.

[2]Breitling R, Sorokin A V, Behnke D. Temperature-inducible gene expression in Bacillus subtilis mediated by the c1857-encoded repressor of bacteriophage lambda. Gene, 1990, 93(1): 35-40.

[3]Gao S, Lu J, Wang T, Xu S, Wang X, Chen K, Ouyang P. A novel co-production of cadaverine and succinic acid based on a thermal switch system in recombinant Escherichia coli. Microbial Cell Factories, 2022, 21 (1): 248.

[4]Choi S Y, Woo H M. CRISPRi-dCas12a: A dCas12a-mediated CRISPR interference for repression of multiple genes and metabolic engineering in cyanobacteria. ACS Synthetic Biology, 2020, 9(9): 2351-2361.

[5]Aguilo-Ferretjans M D M, Bosch R, Puxty R J, et al. Pili allow dominant marine cyanobacteria to avoid sinking and evade predation. Nature Communications, 2021, 12(1): 1857.

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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 PR 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 PR promoter and the TrbcL terminator to construct a recombinant plasmid pS1-ssr1114 (figure 3).

**Table 1: The conditions and process for fusion-PCR**

![](https://static.igem.wiki/teams/5259/experiments/1.png)

![](https://static.igem.wiki/teams/5259/experiments/2.png)

**Table 2: The primers used for fusion-PCR**

![](https://static.igem.wiki/teams/5259/experiments/3.png)

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 PR 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**

![](https://static.igem.wiki/teams/5259/experiments/4.png)

![](https://static.igem.wiki/teams/5259/experiments/5.png)

*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).

![](https://static.igem.wiki/teams/5259/experiments/6.png)![](https://static.igem.wiki/teams/5259/experiments/7.png)

![](https://static.igem.wiki/teams/5259/experiments/8.png)![](https://static.igem.wiki/teams/5259/experiments/9.png)

*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;

![](https://static.igem.wiki/teams/5259/experiments/10.png)![](https://static.igem.wiki/teams/5259/experiments/11.png)

![](https://static.igem.wiki/teams/5259/experiments/12.png)![](https://static.igem.wiki/teams/5259/experiments/13.png)

*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.

![](https://static.igem.wiki/teams/5259/experiments/14.png)

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

![](https://static.igem.wiki/teams/5259/experiments/15.png)

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**

![](https://static.igem.wiki/teams/5259/experiments/16.png)

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.

![](https://static.igem.wiki/teams/5259/experiments/17.png)

*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.

![](https://static.igem.wiki/teams/5259/experiments/18.png)

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

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Results

![](https://static.igem.wiki/teams/5259/results/1.png)![](https://static.igem.wiki/teams/5259/results/2.png)

*Figure 13. Team members collected lake water in the Beiyang Campus of Tianjin University (Left) and filtered the lake water for disinfection for testing (Right).*

![](https://static.igem.wiki/teams/5259/results/3.png)![](https://static.igem.wiki/teams/5259/results/4.png)

*Figure 14. Team members learned how to measure OD750 with spectrophotometer (Left) and diluted the culture medium in order to smear it on solid medium for the estimation of CFU (Right)*

In order to verify whether the engineered *S. elongatus* PCC 7942 cells (transformed with pS1-ssr1114 and pS2-slr0664 plasmids) were successfully conferred with the property that they would be killed by the toxin Slr0664 under an environmental nutrition and temperature condition, we acquired some natural water from a lake at the Beiyang Campus of Tianjin University and inoculated the engineered strain and wild-type strain of *S. elongatus* PCC 7942 cells into the lake water (disinfected by filter) respectively. These inoculated bacteria were then cultivated in the illuminating shaker at 3 different temperatures (25℃, 30℃ and 37℃). Sample from each medium was collected every day to record its OD750, until day 6. At the same time, sample from each medium was also collected to smear the solid medium to estimate the colony forming unit (CFU). The CFU was calculated by the following formula:

CFU/ml = Mean colony number × dilution factor / volume of the medium

According to our OD750 results (Table 5, Figure 15), both engineered and wild-type S. sp. PCC 7942 cyanobacteria showed similar growth curves, when they were cultured in lake water at 37℃. The wild-type strain grew well at 37℃, as well as at 25℃ and 30℃, whereas the engineered strain was not able to grow at all at 25℃ and 30℃. The CFU results (Table 6, Figure 16) further indicated that the wild-type strain maintained a stable CFU across all tested temperatures, and the engineered strain cultured at 37℃ showed stable CFU from day 0 to day 6. However, the CFU of the engineered strain culture at 25℃ and 30℃ declined sharply over this period (at 30℃, the CFU dropped to zero by day 5, while at 25℃, it reached zero by day 4). These results indicate that our engineered cyanobacteria strain can be effectively killed by the toxin at temperatures below 37℃, but can grow normally at 37℃. However, complete elimination required 4-5 days. According to NIH guidelines, the escape frequency of genetically engineered organisms should be lower than 10-8, meaning no more than one bacterium in 108 is allowed to escape into the environment. This suggests that the efficiency of our toxin-antitoxin biocontainment system could meet the standard after 5 days after leakage.

**Table 5: Data of OD750**

![](https://static.igem.wiki/teams/5259/results/5.png)

**Table 6: Data of CFU**

![](https://static.igem.wiki/teams/5259/results/6.png)

![](https://static.igem.wiki/teams/5259/results/7.png)

*Figure 15. (A) Growth results by OD750. The engineered cyanobacteria showed a OD750 curve without any growth from day 0 to day 6 when they were cultured at 25℃ and 30℃. The other groups showed a normal growth curve. (B) Demonstration of engineered cyanobacteria cultivated at 37℃ (Left), 30℃ (Middle), 25℃ (Right) on day 3.*

![](https://static.igem.wiki/teams/5259/results/8.png)

*Figure 16. (A) Growth results by CFU. The CFU of the engineered cyanobacteria cultivated at 25℃ and 30℃ declined dramatically from day 0 to day 6, while CFU of the other groups did not show a significant change in this period. (B) Few colonies found for the Engineered strain than the wild-type strain grown plates.*

For the *pilO* knockdown project, our team have collected cell movement data for both WT and the engineered strains (Figure 17). The results are demonstrated and analyzed in the part of dry lab.

![](https://static.igem.wiki/teams/5259/results/9.png)![](https://static.igem.wiki/teams/5259/results/10.png)

*Figure 17. Team members were preparing for the cyanobacteria samples for microscopy (left) and monitered the mobility of the cyanobacteria under optical microscope.*

As our final goal is to realize a temperature-sensitive knock-out of *pilO* gene similar to what we achieved for the toxin-antitoxin system, namely the crRNA of *pilO* should only be transcribed when the temperature is lower than 37℃. To do so, we planned to insert the gene of the repressor protein lacI behind the PR promoter in order to accomplish a temperature-controlled expression of this repressor in the near future. Then, the promoter PpsbA2 in front of the crRNA gene will be replaced by a promoter sensitive to LacI repressor. Then, in this system, crRNA against *pilO* will be only produced at a temperature below 37℃.

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