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].

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 P_R, 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 P_R promoter [2]. Without the binding of cI857, P_R 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 P_R 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).

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

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).

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.

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.