Part:BBa_K5480009:Design

Design Concept

The Y38-DpnI-TrrnB composite part is a temperature-dependent construct designed to precisely regulate gene expression and control cell fate under specific thermal conditions. This part leverages the Y38 promoter as a temperature-sensitive regulatory element, in combination with the DpnI restriction enzyme, to induce temperature-dependent cell death. To ensure efficient transcription termination and prevent transcriptional read-through, the TrrnB terminator is integrated into the composite part.

Mechanism of Action

At 37°C, the Y38 promoter is activated, leading to the expression of the DpnI gene. The DpnI enzyme recognizes and cleaves specific sites in the E. coli genome, resulting in DNA fragmentation and cell death. The following diagrams illustrate the regulatory mechanism of the Y38 promoter:

Temperature-regulated control of DpnI expression via the Y38 promoter.
At temperatures equal to or above 37°C, the Y38 promoter is activated, leading to the expression of the DpnI gene. The expressed DpnI enzyme cleaves the DNA, ultimately causing cell death. In contrast, at approximately 30°C, the Y38 promoter is inactivated, preventing DpnI expression and allowing cells to survive. This temperature-dependent system enables precise control over cell fate, ensuring cell survival under lower temperatures and self-destruction when temperatures rise above 37°C

Furthermore, the widespread occurrence of DpnI recognition sites in the E. coli genome ensures efficient degradation of DNA by the DpnI enzyme, as shown in the following diagram:

Distribution of DpnI recognition sites across the E. coli genome.
This graph highlights the widespread presence of DpnI recognition sites throughout the E. coli genome, demonstrating the enzyme’s efficiency in cleaving DNA. The abundance of these sites makes DpnI a highly effective tool for DNA fragmentation, ensuring rapid cell death when expressed.

Plasmid Design

The following plasmid map provides an overview of the construction of the Y38-DpnI-TrrnB composite part, highlighting the key functional elements such as the Y38 promoter, the DpnI gene, and the TrrnB terminator. This design ensures precise regulation of gene expression under the appropriate conditions:

Cloning of the Y38-DpnI-TrrnB composite part into the pYBa plasmid.
This plasmid map shows the insertion of the Y38-DpnI-TrrnB composite part into the pYBa backbone. The key functional elements, including the Y38 promoter, DpnI gene, and TrrnB terminator, are positioned for efficient gene expression and regulation within the construct.