Parts
BBa_K5259000 is a plasmid constructed by our team within our iGEM project that aimed to construct a temperature-sensing biocontainment system to induce cyanobacteria “suicide” in non-permissive conditions, such as natural environments with night temperature typically below 30℃, but grow normally under laboratory conditions (37℃). Temperature condition is a unique input signal characterized by its non-invasive nature, good penetrability, low cost, and reversibility which has not been used for environmental biocontainment previously, to our best knowledge. This system includes a toxin-antitoxin system 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].
In order to achieve our goal, we have to find a mechanism to control the expression of ssr1114 by temperature, namely express it at or above 37℃, but stop its transcription at a temperature below 37℃. Therefore, the construction of the plasmid with the part number BBa_K5259000 plays a crucial role in our project.
Figure 1. Scheme of the plasmid pS1-ssr1114 (BBa_K5259000).
This plasmid contains a temperature-sensitive promoter (PR) and the gene of its corresponding repressor protein (cI857) previously found in bacteriophage lambda (λ) [2]. During our project, our team inserted then the antitoxin gene ssr1114 gene behind the PR promoter. 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].
The successful construction of the plasmid BBa_K5259000 (Figure 1), coordinating with the other plasmid BBa_5259001 (including the toxin gene slr0664), helps us to initiate a biocontainment system, in which engineered cyanobacteria stain can grow normally in our lab's incubator at 37℃, but die within 5 days when they were cultured at a temperature lower than 30℃, according the results of our escape experiments conducted under both laboratory conditions and stimulated natural pond conditions in Tianjin, China demonstrated in Figures 2 and 3. The results proved that our system has successfully achieved biocontainment with survival rates below the standard proposed by the National Institute of Health (NIH) guidelines for organisms containing recombinant DNA molecules (10−8).
The highlight of our temperature-sensing system with the part (BBa_5259000) avoids the addition of any exogenous chemical inducer, not only cutting down the cost but also decease any secondary chemical contamination. Additionally, this part (BBa_5259000) not only refers to the biocontainment of cyanobacteria, but can also be applied in other chassis organisms for synthetic biology. It’s no doubt that our system will make a great contribution for the secure development of synthetic biology.
Figure 2. (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 3. (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.
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.