Part:BBa_K5480009

Overview

The Y38-DpnI-TrrnB composite part is designed to induce cell death at 37°C via the expression of the DpnI restriction enzyme. The construct features a temperature-sensitive Y38 promoter, which activates the expression of the DpnI gene at 37°C, leading to DNA cleavage and cell death. At 30°C, the Y38 promoter is inactive, preventing DpnI expression and allowing the cells to survive. This composite part offers an effective solution for projects that require precise control over cell fate, particularly in applications related to biosafety and secure information transmission.

Components

Y38 Promoter: A temperature-sensitive promoter that activates expression at 37°C and is inactivated at 30°C. DpnI Gene: Encodes the DpnI restriction enzyme, which recognizes and cleaves specific DNA sequences. TrrnB Terminator: Ensures efficient termination of transcription and prevents transcriptional interference.

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:

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       <figcaption>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.</figcaption>
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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:

< img src="https://static.igem.wiki/teams/5480/part/part-2.png" width="300" height="300" />

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:

< img src="https://static.igem.wiki/teams/5480/part/part-3.png" width="300" height="300" />

Cloning of the Y38-DpnI-TrrnB composite part into the pYB1a plasmid. This plasmid map shows the insertion of the Y38-DpnI-TrrnB composite part into the pYB1a 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.

Applications

Biosafety: This part is ideal for use in applications where it is crucial to prevent the spread of biological materials, such as controlling live-cell biosensors or securely handling sensitive biological data. When the environmental temperature reaches 37°C, cells self-destruct due to DpnI expression, providing an effective biosafety mechanism.

Secure Information Transmission: By embedding sensitive information within cells, this part can ensure that once the information is decoded, the cells will self-destruct at 37°C, preventing information leakage.

Experimental Validation

We have experimentally validated the temperature sensitivity of the Y38 promoter and the efficacy of DpnI expression. The following images demonstrate the comparison between cell death at 37°C and survival at 30°C:

< img src="https://static.igem.wiki/teams/5480/part/part-4.png" width="300" height="300" />

Petri dishes demonstrating the effects of DpnI expression at different temperatures. The left image shows the plate at 37°C, where DpnI expression is activated, leading to cell death. The right image shows the plate at 30°C, where the Y38 promoter is inactive, allowing cell survival. This confirms the temperature-sensitive control of cell fate through the Y38-DpnI-TrrnB composite part.