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Introduction: Thermosynechococcus elongatus BP1 is a thermophilic strain of cyanobacteria that has an optimum growth at 57°C, and according to previous analysis by Yamaoka et al, T elongatus BP1 cannot survive at a temperature below 30°C. This suggests that the thermophilic property of this strain may be used as a natural biosafety feature to limit the spread of genetically engineered (GE) organisms in the environment if physical containment fails. Objective: To further explore the growth and survivability range of T elongatus BP1, we report a growth and survivability assay of wild-type and GE T elongatus BP1 strains under different conditions. Methods: Wild-type and GE T elongatus BP1 cultures were prepared and incubated in the laboratory (high temperatures and constant light source) and greenhouse conditions (lower/varied temperatures and sunlight) for 4 weeks. The cell density was monitored weekly by measuring the optical density at 730 nm (OD730). To assess the survivability, a sample of each culture was added to fresh media, placed in laboratory conditions (42.2°C and 30 µE m–2 s–1) in multi-well plates and observed for growth for up to three weeks. Lastly, the number of viable cells were determined by plating a diluted sample of the culture on solid media and counting colony-forming units (CFU) after 1 day, 2 weeks and 4 weeks of incubation in laboratory or greenhouse conditions. Results: Our experimental results demonstrated that growth was hindered but that the cells did not entirely die within 2 to 4 weeks at warm temperatures (31.42°C-36.27°C). The study also showed that 2 weeks of exposure to cool temperature conditions (15.44°C-25.30°C) was enough to cause complete death of GE T elongatus BP1. However, it took 2 to 4 weeks for the wild-type T elongatus BP1 cells to die. Conclusion: This study revealed that the thermophilic feature of the T elongatus BP1 may be used as an effective biosafety mechanism at a cool temperature between 15.44°C and 25.30°C but may not be able to serve as a biosafety mechanism at warmer temperatures.

Genetically engineered (GE) cyanobacteria are an attractive photosynthetic platform for sustainable biofuels and bioproducts. A number of biofuels such as ethanol, isobutanol, and 1‐butanol have been experimentally produced using GE cyanobacteria. Even though this approach is promising, there are still questions regarding the biosafety of these GE organisms. What happens to the plasmids that are used for introducing transgenes into the cyanobacterial cells? Studies have shown that E. coli can transfer plasmids into cyanobacteria. We are interested in knowing if the reverse direction is possible? Furthermore, can the transgenes be horizontally transferred into other organisms after they are already integrated into the genomic DNA of the cyanobacteria? These questions need to be addressed before this approach can be commercialized. In order to answer these questions, we conducted an experiment by co‐incubating wild‐type E. coli DH5α with different sets of GE Thermosynechococcus elongatus BP1 carrying either plasmid transgenes or solely integrated transgenes. The transfer event was monitored using selection markers. Our results showed that the transgenes in the form of plasmids were transferred from these GE cyanobacteria into wild‐type E. coli DH5α even without the help of a nitrocellulose membrane while there was no detectable transfer event for the integrated transgenes observed so far. In addition, the frequency of the plasmid transfer event appeared to decrease gradually over time. These results suggested that even though GE cyanobacteria carrying integrated transgenes possess a more permanent change in the genomic DNA, the bio‐risk of these GE organisms in the context of gene sharing is less severe than plasmids‐carrying GE cyanobacteria.Support or Funding InformationThis work is supported by Biotechnology Risk Assessment Grant Program competitive grant award no. 2016‐33522‐25624 from the U.S. Department of Agriculture to JWL and LHG.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.