Results

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).

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

Table 6: Data of CFU

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.

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.

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 P_R 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℃.