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To verify the successful amplification of mazE and mazF genes, we performed PCR using specific primers targeting these two genes. The PCR reaction was carried out with a standard protocol, followed by agarose gel electrophoresis to confirm the size of the amplified products. The expected length for mazE was approximately 300 bp, and for mazF, it was around 400 bp.After running the PCR products on a 1% agarose gel, clear bands were observed at the expected sizes for both mazE and mazF (Figure 2).  
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To verify the successful amplification of mazE and mazF genes, we performed PCR using specific primers targeting these two genes. The PCR reaction was carried out with a standard protocol, followed by agarose gel electrophoresis to confirm the size of the amplified products. The expected length for mazE was 252 bp, and for mazF, it was 336 bp.After running the PCR products on a 1% agarose gel, clear bands were observed at the expected sizes for both mazE and mazF (Figure 2).  
  
 
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<p style="font-size: 98%; line-height: 1.4em;">Figure 2. Agarose gel electrophoresis of mazE and mazF.The expected band sizes of 252 bp for mazE, 336 bp for mazF and the maker used was 2kb.</p >
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<p style="font-size: 98%; line-height: 1.4em;">Figure 2. Agarose gel electrophoresis of mazE and mazF.The expected band sizes of 252 bp for mazE, 336 bp for mazF and the marker used was 2kb.</p >
 
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To validate the functionality of the suicide system, the engineered E. coli BL21 strain with the rhamnose-inducible suicide system (BL21/PBAD-mazE-PRha) was inoculated into LB medium and cultured overnight at 37°C. The next day, the overnight culture was diluted 1:50 into fresh M9 medium containing 50 μg/mL ampicillin, 1% rhamnose, 0.2% arabinose, and 0.4% glucose. The OD600 was continuously monitored using a microplate reader to evaluate the impact of the suicide system on bacterial growth. The results showed that both the BL21 and BL21/PBAD-mazE-PRha strains (with 1% L-arabinose) grew normally during the 8-hour period, reaching an OD600 value of approximately 0.7, indicating that these strains were unaffected by the suicide system. However, in the absence of L-arabinose, the BL21/PBAD-mazE-PRha strain exhibited a significant decline in growth after 3 hours, indicating that the suicide system had been activated, leading to cell death (Figure 3).
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To validate the functionality of the suicide system, the engineered E. coli BL21 strain with the rhamnose-inducible suicide system (BL21/PBAD-mazE-PRha) was inoculated into LB medium and cultured overnight at 37°C. The next day, the overnight culture was diluted 1:50 into fresh M9 medium containing 50 μg/mL ampicillin, 1% rhamnose, 0.2% arabinose, and 0.4% glucose. The OD600 was continuously monitored using a microplate reader to evaluate the impact of the suicide system on bacterial growth. The results showed that both the BL21 and BL21/PBAD-mazE-PRha strains (with 1% L-rhamnose) grew normally during the 8-hour period, reaching an OD600 value of approximately 0.7, indicating that these strains were unaffected by the suicide system. However, in the absence of L-rhamnose, the BL21/PBAD-mazE-PRha strain exhibited a significant decline in growth after 3 hours, indicating that the suicide system had been activated, leading to cell death (Figure 3).
  
 
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Latest revision as of 11:22, 1 October 2024


Rha-MazTox

Rha-MazTox is a rhamnose-inducible toxin-antitoxin suicide system. In this construct, the rhamnose promoter (Rha) controls the expression of the mazF toxin gene, which encodes an mRNA endonuclease that inhibits bacterial growth by cleaving cellular mRNA, leading to cell death. Simultaneously, mazE, the antitoxin gene, is expressed under a separate control and neutralizes the toxic effects of MazF under normal conditions. When rhamnose is present, it induces mazF expression without sufficient mazE protection, activating the suicide mechanism.

Description

Rha-MazTox (BBa_K5084020) is a rhamnose-inducible controllable suicide system designed to prevent bacterial overgrowth or contamination by regulating the expression of toxin and antitoxin genes. The system utilizes the toxin gene mazF and the antitoxin gene mazE, controlled by two different inducible promoters, providing precise control over bacterial survival based on environmental conditions.

Usage and Biology

To construct a controllable suicide system, the antitoxin gene mazE and toxin gene mazF were synthesized and cloned into the pSB1A3 plasmid. The antitoxin gene mazE was placed downstream of the rhamnose-inducible promoter PRha and RBS B0034, regulated by rhamnose. The toxin gene mazF was positioned downstream of the arabinose-inducible promoter PBAD and regulated by arabinose. After sequencing verification, the recombinant plasmid Rha-MazTox (BBa_K5084020) was transformed into E. coli BL21 to validate the functionality of the suicide system.

FFigure 1. The gene circuit of Rha-MazTox.

Characterization

To verify the successful amplification of mazE and mazF genes, we performed PCR using specific primers targeting these two genes. The PCR reaction was carried out with a standard protocol, followed by agarose gel electrophoresis to confirm the size of the amplified products. The expected length for mazE was 252 bp, and for mazF, it was 336 bp.After running the PCR products on a 1% agarose gel, clear bands were observed at the expected sizes for both mazE and mazF (Figure 2).

Figure 2. Agarose gel electrophoresis of mazE and mazF.The expected band sizes of 252 bp for mazE, 336 bp for mazF and the marker used was 2kb.

To validate the functionality of the suicide system, the engineered E. coli BL21 strain with the rhamnose-inducible suicide system (BL21/PBAD-mazE-PRha) was inoculated into LB medium and cultured overnight at 37°C. The next day, the overnight culture was diluted 1:50 into fresh M9 medium containing 50 μg/mL ampicillin, 1% rhamnose, 0.2% arabinose, and 0.4% glucose. The OD600 was continuously monitored using a microplate reader to evaluate the impact of the suicide system on bacterial growth. The results showed that both the BL21 and BL21/PBAD-mazE-PRha strains (with 1% L-rhamnose) grew normally during the 8-hour period, reaching an OD600 value of approximately 0.7, indicating that these strains were unaffected by the suicide system. However, in the absence of L-rhamnose, the BL21/PBAD-mazE-PRha strain exhibited a significant decline in growth after 3 hours, indicating that the suicide system had been activated, leading to cell death (Figure 3).

Figure 3. Effect of rhamnose-induced suicide system on bacterial growth.

Potential application directions

The Rha-MazTox system has several potential applications, primarily in the controlled regulation of bacterial survival. It can be used for biosafety and biocontainment to prevent genetically modified organisms from escaping into the environment when specific inducers are absent. In industrial fermentation, it can help limit bacterial overgrowth to ensure optimal production yields. Additionally, it has applications in medical or environmental fields, where precise control of bacterial populations is required, such as targeted elimination or containment. This system offers a safe and reliable solution for effective management of bacterial populations.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 195
    Illegal BamHI site found at 1689
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1524
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 1506

References

Engelberg-Kulka H, Hazan R, Amitai S. mazEF: a chromosomal toxin-antitoxin module that triggers programmed cell death in bacteria[J]. Journal of cell science, 2005, 118(19): 4327-4332. Guzman L M, Belin D, Carson M J, et al. Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter[J]. Journal of bacteriology, 1995, 177(14): 4121-4130. Giacalone M J, Gentile A M, Lovitt B T, et al. Toxic protein expression in Escherichia coli using a rhamnose-based tightly regulated and tunable promoter system[J]. Biotechniques, 2006, 40(3): 355-364.