Part:BBa_K4833024

This sequence expresses two enzymes crucial for DAP synthesis in the presence of Arabidose.

araC::pBAD::murA::asd

MurA and asd genes are placed under the control of the pBAD promoter, allowing for arabinose-induced expression of enzymes essential for peptidoglycan synthesis and maintenance.

Background

Bacterial survival and cell wall integrity are crucial for maintaining bacterial function, especially in strains used for therapeutic applications. Two key genes involved in bacterial cell wall biosynthesis are murA and asd. MurA plays an essential role in the peptidoglycan synthesis pathway, which is critical for maintaining the strength and structure of the bacterial cell wall. Asd is necessary for the biosynthesis of diaminopimelic acid (DAP), an essential component of the bacterial cell wall that enables the formation of cross-links within peptidoglycan layers. Without the expression of murA and asd, bacteria lose the ability to sustain their cell walls, leading to cell lysis. To regulate the expression of these genes in a controllable manner, the araC gene and the pBAD promoter system are employed. The araC gene encodes the AraC protein, a transcriptional regulator that activates the pBAD promoter in the presence of arabinose. This inducible system allows for precise control over gene expression, making it ideal for applications where conditional bacterial survival is needed, such as in attenuated bacterial strains used for tumor-targeting therapies.

Usage and Biology

The araC::pBAD::murA::asd composite part is designed to conditionally rescue the cell wall synthesis in engineered Salmonella strains that lack functional murA and asd genes in their genome. By placing these two essential genes under the control of the pBAD promoter, this system ensures that bacterial survival is dependent on the presence of arabinose in the environment. This allows for tight regulation of bacterial survival, enabling bacterial lysis in the absence of arabinose, a feature that enhances the safety of engineered bacterial therapies used in tumor targeting. In the presence of arabinose, the araC gene activates the pBAD promoter, inducing the expression of murA and asd. This leads to the restoration of bacterial cell wall biosynthesis, allowing the bacteria to survive and function normally. When arabinose is absent, the pBAD promoter remains inactive, resulting in the lack of murA and asd expression, and consequently bacterial lysis due to the inability to maintain cell wall integrity. This conditional lysis mechanism makes the composite part suitable for applications where controlled bacterial death is essential, such as during tumor therapy, where bacteria should only survive and replicate in specific conditions, like the tumor microenvironment.

Design

The araC::pBAD::murA::asd composite part consists of the arabinose-inducible pBAD promoter, regulated by the araC gene, driving the expression of two essential genes, murA and asd, required for bacterial cell wall synthesis. The system ensures bacterial survival only in the presence of arabinose, allowing for controlled and safe proliferation of the bacteria. The sequence was incorporated into the plasmid pSilencer-CLDN6 for experimental validation.

Figure 1. plasmid pSilencer-CLDN6.

Validation of Regulated Delayed Lysis System

To validate the functionality of the delayed lysis system, we transformed the χ11803 strain with the plasmid containing the part. The bacteria were cultured in LB, LB with arabinose, and LB with carbenicillin to observe growth. The results showed that bacterial growth occurred only in the arabinose-containing LB medium, confirming that the system is functional and arabinose-dependent.

Figure 2. Growth of bacteria in LB, LB with arabinose, and LB with carbenicillin media.

Additionally, when plated on LB and arabinose-containing plates, only the arabinose plates showed bacterial growth, further supporting the delayed lysis system's effectiveness.

Figure 3. Growth of bacteria on LB and LB with arabinose plates.

Conclusion and Outlook

The araC::pBAD::murA::asd system has demonstrated effective regulation of bacterial survival, with murA and asd expression strictly dependent on the presence of arabinose. This part provides a safe and controllable method for bacterial lysis, which is especially useful in therapeutic applications where bacterial clearance is essential. Future applications may include using this system in cancer-targeting bacterial therapies or other biomedical interventions where precise control of bacterial survival and lysis is required. Additionally, this system may be adapted for use with other bacterial strains or therapeutic payloads to enhance its utility in clinical or experimental settings.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1128
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1067
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 909
  Illegal AgeI site found at 1275
  Illegal AgeI site found at 1867
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 2893
  Illegal SapI site found at 891