Latest revision as of 03:32, 2 October 2024

Alkane-responsive suicide system

This composite part is designed as an alkane-responsive suicide system. The mazE gene, encoding an antitoxin protein, is placed downstream of the PalkB promoter, which is induced by the presence of alkanes. This ensures that mazE is expressed only in alkane-rich environments. Meanwhile, the mazF gene, encoding a toxin protein, is positioned downstream of the PBAD promoter, which controls its expression. This system allows the bacteria to survive in the presence of alkanes but triggers cell death when alkanes are absent, ensuring biosafety.

Usage and Biology

The BBa_K5468009 part is designed to address biosafety concerns by creating an alkane-responsive suicide system in E. coli. This system ensures that the genetically modified bacteria can survive only in environments contaminated with alkanes, such as petroleum spills, and will self-destruct in clean environments.

Biological Function

The part integrates a toxin-antitoxin system (MazF/MazE) controlled by two different promoters: the alkane-inducible PalkB promoter and the arabinose-inducible PBAD promoter. The PalkB promoter is activated in the presence of alkanes, inducing the expression of mazE, which encodes the antitoxin MazE. This protein neutralizes the toxic effects of MazF, allowing the engineered bacteria to survive in alkane-contaminated environments.

Conversely, mazF, the toxin gene, is controlled by the PBAD promoter. When alkanes are absent and the PalkB promoter is inactive, MazF is expressed under arabinose induction and kills the bacteria by cleaving mRNA. This ensures that the bacteria cannot survive outside the alkane-contaminated area, providing an effective biocontainment strategy.

Figure 1. Schematic diagram of the suicide system

Mechanism

Presence of Alkanes: The PalkB promoter is activated, and mazE is expressed, producing the MazE protein, which neutralizes MazF, ensuring bacterial survival. Absence of Alkanes: The PalkB promoter is inactive, and mazE is not produced. If arabinose is present, mazF is expressed from the PBAD promoter, leading to bacterial cell death as MazF cleaves mRNA, halting protein synthesis.

Applications

This part is particularly useful in environmental biotechnology, especially for the bioremediation of petroleum-contaminated sites. The alkane-responsive feature ensures that the bacteria operate safely within the contaminated environment, and the suicide mechanism prevents their escape into non-polluted ecosystems, mitigating risks associated with genetically modified organisms (GMOs).

Comparison with Existing Systems

Compared to other biocontainment strategies, such as temperature-sensitive kill switches or antibiotic-based systems, the alkane-responsive mechanism offers a more natural and field-relevant control system. This design aligns with sustainable environmental practices, as it eliminates the need for external chemicals like antibiotics or temperature control, making it suitable for real-world applications.

Characterization

Construction of the Suicide System

Objective and Methods

To construct the suicide system, sequences encoding PalkB, mazF, and mazE were synthesized and cloned into the pSB1A3 vector using XbaI and SpeI restriction sites. In this system, mazF, which encodes a toxin, is constitutively expressed, while the antitoxin gene mazE is placed downstream of the PalkB promoter. The PalkB promoter is induced by alkanes, meaning that the presence of alkanes will trigger the expression of mazE, neutralizing the toxic effect of mazF.

Figure 2. Gel Electrophoresis of Suicide System Components (PalkB, mazF, mazE)

Results and Conclusion

The gel electrophoresis results confirm the successful amplification of the following gene sequences: PalkB: 2966 bp mazF: 336 bp mazE: 252 bp These results indicate successful cloning of the key components of the suicide system into the pSB1A3 vector. This system ensures that in the absence of alkanes, mazF will kill the host cells, preventing their survival, while in the presence of alkanes, mazE will be expressed, allowing the cells to remain viable. Such a system can be used as a biocontainment strategy in environmental applications where it is important to control the survival of engineered microorganisms.

Alkane Promoter Test

Objective and Methods

To test the functionality of the alkane-inducible promoter, sequences encoding AlkS and PalkB were synthesized and cloned into the pSB1A3 vector using XbaI and SpeI restriction sites. These were placed upstream of the mRFP fluorescent protein gene. The expression of the transcription factor AlkS was controlled by the constitutive J23100 promoter. The recombinant plasmid was transformed into E. coli DH5α cells. The alkane biosensor strains were inoculated into LB medium at a 1:100 ratio and cultured overnight at 37°C. The next day, the culture was diluted 1:50 into fresh M9 medium supplemented with 50 μg/mL ampicillin and 10 g/L glucose. Solutions of n-dodecane dissolved in 1% Tween80 ethanol were prepared at a concentration of 200 mg/L and stirred thoroughly. Different concentrations of n-dodecane solution were added to the culture medium and induced at 37°C for 20 hours. After incubation, 1 mL of the culture was collected, and OD600 and fluorescence values were measured (excitation wavelength: 584 nm, emission wavelength: 607 nm) using a plate reader. The fluorescence was normalized by calculating the fluorescence/OD600 ratio.

Figure 3. Alkane Promoter Test Using mRFP Fluorescence

Results and Conclusion

As shown in the figure above, the fluorescence/OD600 ratio increased with higher concentrations of n-dodecane, indicating that the alkane sensor was successfully activated. This result confirms that the PalkB promoter effectively induces expression in the presence of n-dodecane, demonstrating its potential as a biosensor for detecting alkane levels.

Suicide system test

Objective and Methods

The objective of this experiment was to test the functionality of the alkane-inducible suicide system in E. coli BL21 engineered strains. The engineered system included the toxin gene mazF under the control of the PBAD promoter and the antitoxin gene mazE downstream of the PalkB promoter, which is induced by alkanes. After cloning and verification of the recombinant plasmid, it was transformed into E. coli BL21. To evaluate the effectiveness of the system, the engineered strains were first grown overnight in LB medium at 37°C. The next day, cultures were diluted into M9 medium supplemented with ampicillin, glucose, 0.02% arabinose, and varying concentrations of n-dodecane (alkane). OD600 values were measured to monitor bacterial growth and the functionality of the suicide system under different conditions.

Figure 4. Suicide system test

Results and Conclusion

The results, as shown in the image, demonstrate that the engineered strain BL21-PBAD-MazF showed inhibited growth in the presence of 0.02% arabinose due to the expression of the MazF toxin. However, in the presence of n-dodecane, the PalkB-induced expression of MazE (the antitoxin) partially neutralized the toxic effects of MazF, allowing for moderate bacterial growth. The highest growth recovery was observed when both n-dodecane and 0.02% arabinose were present, indicating that the suicide system can be effectively controlled by the presence of alkanes. The control strain (BL21 without the suicide system) grew normally, showing the highest OD600 values. This experiment confirms the functionality and controllability of the engineered suicide system, with potential applications in biocontainment.

Potential Application Directions for BBa_K5468009: Alkane-Responsive Suicide System

1. Bioremediation of Petroleum-Contaminated Environments The BBa_K5468009 system is ideally suited for use in the bioremediation of oil spills and other petroleum-contaminated environments. By engineering bacteria that can survive and degrade hydrocarbons only in the presence of alkanes, this system ensures that the microorganisms will thrive where they are most needed—within the polluted environment. Once the contamination is cleared or significantly reduced, the absence of alkanes triggers the suicide mechanism, ensuring that the engineered strain does not persist in clean ecosystems.

2. Biosensors for Environmental Monitoring The alkane-inducible PalkB promoter can be repurposed as a biosensor to detect the presence of alkanes or hydrocarbons in the environment. When integrated with a reporter gene such as GFP or RFP, this system could provide a visual or measurable signal in response to alkane presence, allowing for real-time monitoring of petroleum contamination levels. This can be valuable in detecting leaks from oil storage facilities, pipelines, or natural reserves.

3. Industrial Waste Treatment Many industrial processes, especially in the petrochemical industry, generate waste streams containing alkanes and other hydrocarbons. The alkane-responsive system could be employed in bacteria designed to treat such waste. This would enable safe degradation of harmful compounds within controlled environments like bioreactors, where the bacteria would function until the alkanes are fully processed, after which the suicide system would prevent bacterial escape into the environment.

4. Biocontainment of Engineered Microorganisms in Oil Fields In oil extraction and processing, microbial enhanced oil recovery (MEOR) techniques use bacteria to increase oil yield. By integrating the BBa_K5468009 suicide system, it would be possible to control these engineered microorganisms, ensuring they survive only within oil fields where alkanes are present. This adds a layer of biosafety to the MEOR process, preventing the unintended spread of engineered microbes into surrounding environments.

5. Controlled Release of Engineered Bacteria for Hydrocarbon Degradation Engineered microbes designed for hydrocarbon degradation in environments such as marine ecosystems or soil can be equipped with the BBa_K5468009 part to ensure they only survive as long as they are performing their intended function. Once hydrocarbons are no longer present in detectable levels, the suicide system would trigger, ensuring no lingering impact on the local microbial community, thus preventing ecological imbalance.

6. Smart Biocontainment Systems for Synthetic Biology Applications This part provides a foundational tool for synthetic biology applications that require precise control of genetically modified organisms (GMOs). By incorporating BBa_K5468009 into synthetic microbial consortia, researchers can create GMOs that are programmed to self-destruct after completing their task, such as environmental cleanup or chemical synthesis, thereby addressing public and regulatory concerns over the release of GMOs.

Reference：

1.Hayes, F. (2003). Toxins-antitoxins: plasmid maintenance, programmed cell death, and cell cycle arrest. Science, 301(5639), 1496-1499.

2.Chikere, C. B., Okpokwasili, G. C., & Chikere, B. O. (2011). Monitoring of microbial hydrocarbon remediation in the soil. 3 Biotech, 1(3), 117-138.

3.van Beilen, J. B., Panke, S., Lucchini, S., Franchini, A. G., Rothlisberger, M., & Witholt, B. (2001). Analysis of Pseudomonas putida alkane degradation gene clusters and flanking insertion sequences: evolution and regulation of the alk genes. Microbiology, 147(6), 1621-1630.

4.Wright, O., Stan, G. B., & Ellis, T. (2013). Building-in biosafety for synthetic biology. Microbiology, 159(7), 1221-1235.

5.Youssef, N., Elshahed, M. S., & McInerney, M. J. (2009). Microbial processes in oil fields: culprits, problems, and opportunities. Advances in Applied Microbiology, 66, 141-251.

Sequence and Features