Contribution

Ammonia responsive promoter mutants，glnAp2 B6,B7,C6,D5

To ensure the modified Escherichia coli strains are viable in a septic tank environment designed for "ammonia suicide," we needed to adapt their response to varying ammonia nitrogen concentrations. We reached out to the Beijing Municipal Wastewater Treatment Center in China, requesting data on the physicochemical indicators of sewage in residential septic tanks. We were fortunate to receive information regarding ammonia nitrogen levels in Changping District, Beijing, for the year 2022. These levels ranged from 150 mg/L to 450 mg/L, with bi-monthly testing, averaging approximately 298.7 mg/L over 24 tests conducted in 2022. Armed with this data, we initiated our experiments.

Using the original glnAP2 promoter sequence as a starting point, we employed error-prone PCR amplification with the Beyotime QuickMutation™ Gene Random Mutagenesis Kit. This process generated a library of mutant sequences, which we then combined through homologous recombination with the mCherry fluorescent protein and the pZe plasmid backbone. The result was a fusion plasmid containing the entire mutation library, known as pZe-glnAP2(random)-mCherry.

Our selection criteria were as follows: Groups 0 and 150 displayed normal fluorescence, while groups 300 and 450 exhibited weak fluorescence.

For each corresponding bacterial strain, we extracted plasmid DNA and reserved 5 μL for future use. The remaining DNA was sent to Huada Company for sequencing.

In practical terms, it's important to note that random mutagenesis lacks specificity in sequence modification, often requiring substantial time and continuous screening to obtain the desired sequences. Fortunately, after a period of screening, BUCT acquired a sample referred to as "B6 B7 C6 D5," consisting of four samples that met the practical application requirements.You can search them on the part page with the following numbers，glnAP2 B6 BBa_K4644005; glnAP2C6 BBa_K4644007 ; glnAP2B7 BBa_K4644008; glnAP2D5 BBa_K4644009.we screened about 1600 samples in total, and finally found these four samples in all mutated samples

Indeed, the promoter samples after random mutation exhibited a noteworthy decrease in intensity as the ammonia nitrogen concentration increased in our practical conditions. This observation serves as a crucial foundation for our efforts to construct a suicide system that can operate effectively under various ammonia nitrogen concentrations as its core feature.

Ammonia responsive kill switch

To meet our biosafety requirements, we integrated the bsrG-asrG toxin-antitoxin system, specifically the Type I toxin-antitoxin system bsrG/asrG(SR4), which originates from Bacillus subtilis strain A-5. Within this system, the antitoxin, asrG, functions as a cis-encoded regulatory RNA that neutralizes the action of the BsrG toxin. AsrG accomplishes this by preventing toxin expression, promoting the degradation of toxin mRNA, and inhibiting its translation.

To control the expression of the suicide system, we harnessed the transcriptional regulator TtgR, sourced from Pseudomonas putida NBRC 14164, which belongs to the TetR family of transcriptional repressors. TtgR has the capability to inhibit the transcription of the TtgABC operon and its own transcription, thereby regulating the efflux of harmful chemicals from bacterial cells through efflux pumps. Importantly, TtgR's ligand-binding domain is quite versatile, allowing it to bind to a wide variety of structurally diverse ligands. You can find it in the BBa_K4644016

With these components in mind, we designed a gene circuit for the suicide system, as illustrated below:

In the human intestinal tract, which typically represents a low-nitrogen environment, the expression of glnAP2 remains relatively high. During this state, the activity of the PttgA promoter is actively repressed by TtgR. To account for possible leakage expression, we have designed the system to co-express the antitoxin of bsrG, ensuring its expression level is significantly higher than that of the toxin. In this configuration, the bacteria can thrive and function normally.

However, once the bacteria find themselves in a septic tank, which is characterized by a high-nitrogen environment, the expression of glnAP2 is inhibited. This inhibition releases the repression on the PttgA promoter, leading to the expression of the toxin. Consequently, the bacteria will undergo programmed cell death, resulting in their demise. This design allows for precise control over the bacteria's survival in different environmental conditions.

The establishment of this suicide system can provide samples and possibilities for the biosafety system of intestinal probiotics in the future. We sincerely hope that the iGEM team in the future can truly benefit from it!