Part:BBa_K5300041
PnuoA-mCherry-RBS-vapC-Ter-PnifH-vapB-PglnK-sgRNA-mCherry-tracrRNA-PnuoA-Cas12k-double Terminator
The CRISPRi system is available to the suicide circuit. We used the Cas12k protein, which binds reversibly to target sequences under the guidance of sgRNA. When external signals reach a certain threshold, Cas12k inhibits transcription of corresponding sequence. Cas12k lacks cleavage activity, so it physically blocks transcription without cutting the target DNA sequence. As the intracellular concentration of sgRNA decreases, the bound Cas12k protein detaches from the target sequence, allowing normal transcription to resume when continuous repression is no longer needed.
We also involved the endogenous toxin-antitoxin (TA) system of the chassis Sinorhizobium fredii CCBAU45436.
In this composite part, vapC encodes the toxin, and vapB encodes the antitoxin. The expression of these genes is regulated by the promoters nifH and glnK. Under high oxygen conditions, expression of vapB is suppressed, and under low nitrogen conditions, the CRISPRi system is activated to suppress expression of vapC. When Sinorhizobium escapes the symbiotic environment and enters a high-nitrogen, high-oxygen external environment, vapC will be expressed normally, while vapB’s expression is suppressed by the high-oxygen conditions. This leads to toxin levels exceeding the tolerance threshold of the antitoxin, ultimately triggering the self-destruction of Sinorhizobium. This design prevents uncontrolled growth of Sinorhizobium in external environments, ensuring the system's safety and precision.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 2013
Illegal SpeI site found at 1272
Illegal PstI site found at 513 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 2013
Illegal NheI site found at 1005
Illegal SpeI site found at 1272
Illegal PstI site found at 513 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 2013
Illegal BglII site found at 1915 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 2013
Illegal SpeI site found at 1272
Illegal PstI site found at 513 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 2013
Illegal SpeI site found at 1272
Illegal PstI site found at 513
Illegal NgoMIV site found at 1394
Illegal AgeI site found at 5
Illegal AgeI site found at 1601
Illegal AgeI site found at 3797 - 1000COMPATIBLE WITH RFC[1000]
Plasmid construction and validation
We first amplified the glnK promoter, nifH promoter, vapC and vapB from the genome, amplified mCherry from the pRJPaph-mChe plasmid and designed the sgRNAs. We screened the loci from the genome, and amplified UP-feo-suicide and DOWN-feo-suicide by PCR. Then we performed seamless cloning to link them to pJQ200SK (Figure 4-1-1).
The recombinant plasmid was transferred into E. coli, and the correct recombinant plasmid was verified by colony PCR.
Triparental mating and validation
The verified correct colonies were inoculated into gentamicin-resistant LB liquid medium under shaking conditions and cultured, triparental mating was performed using Helper, Sinorhizobium fredii CCBAU45436, and two screenings were performed. The single colonies obtained from the second screening were subjected to colony PCR utilizing the universal primer M13F/R. Colony PCR demonstrated that the plasmid successfully underwent homologous recombination with Sinorhizobium fredii CCBAU45436, and the suicide circuit was successfully introduced into chassis. It is noteworthy that the module showed a different phenotype from the wild type when cultured for more than 3 d after introduction, i.e., the colonies became transparent teardrop-shaped.
Fluorescence intensity verification
We utilized its fluorescence expression intensity to verify whether the suicide module functioned properly. We set up four sets of experiment with high nitrogen and high oxygen, low nitrogen and high oxygen, high nitrogen and low oxygen, as well as low nitrogen and low oxygen for verification. Subsequently, we used M9 medium without nitrogen source ,then added different concentrations of ammonium chloride to form nitrogen concentration gradient. We added petroleum jelly after boiling the medium to isolate medium against atmosphere, creating anaerobic conditions. The OD value of the bacterial solution was determined after a period of shaking and incubation, and the fluorescence intensity of the bacterial solution was determined using a fluorescence spectrophotometer (Figure 4-3-1).
In a low oxygen environment, different nitrogen concentrations significantly affect the expression intensity of fluorescent proteins. This is consistent with our design. When changed to high oxygen environment, the change of nitrogen concentration could not significantly affect the fluorescent intensity, which may be caused by the environmental instability due to the expression of toxin. At the same time, we can also find that the fluorescent intensity of the bacterial solution cultured in high oxygen environment was higher than that in low oxygen environment, which may be related to the concentration of the bacterial solution fluctuates more in high oxygen environment by the change of toxin.
Change in OD value of bacterial solution
In order to further verify whether the circuit leads to the death of the bacteria, we selected the strain with suicide circuit plasmid with OD value of 1, added different concentrations of NH4Cl, and investigated the changes of OD values after same interval under hyperoxic conditions (Figure 4-4-1).
Obviously, we found that after a period of time, the OD value of the bacterial solution with additional nitrogen source showed a decreasing trend. And the decreasing trend was more obvious with the increase of the concentration of nitrogen source. Under this experimental condition, it can be considered that our suicide circuit is regulated by nitrogen concentration and the overall pathway construction is successful.
Phenotype validation
In the process of triparental mating, we found that the correct exchanged strains did not express significant trait changes in the medium used for the first screening, whereas the phenotype of hyaline colonies appeared in the medium used for the second screening. We believe that the suicide module played a role in causing the strain to accumulate too much toxin and die. We therefore re-cultured the strain with the suicide module in nitrogen-free M9 medium shaking conditions. The cultured bacteria were spread on TY solid medium to observe their growth (Figure 4-5-1).
The toxin-antitoxin system functions under high nitrogen and high oxygen conditions, and the excess toxin content leads to colony death. We were able to observe that colonies from three-day cultures showed a milky white color, while colonies from five-day cultures showed a transparent color. This is the most direct evidence that the suicide circuit was successfully constructed.
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