Part:BBa_K5480009:Experience

The Y38-DpnI-TrrnB composite part is designed to precisely control gene expression and induce programmed cell death through temperature-dependent regulation. This part features the temperature-sensitive Y38 promoter, which activates the expression of the DpnI restriction enzyme at 37°C, leading to DNA fragmentation and cell death. At 30°C, the Y38 promoter is inactive, allowing cell survival. Below is a summary of the experiments conducted to verify the function of this part, along with key experimental results.

Construct Verification

First, the pY38a-DpnI plasmid was validated through PCR amplification and enzyme digestion. The following gel electrophoresis image shows the expected sizes of the DpnI gene (805 bp), Y38 promoter (1488 bp), and vector (2183 bp), confirming the successful amplification of each component.

Gibson Assembly Reaction

To assemble the pY38a-DpnI plasmid, we utilized the Gibson assembly method. The table below lists the components and their volumes used in the assembly reaction. This step ensured the efficient ligation of the Y38 promoter, DpnI gene, and the vector.

Components and volumes used for the Gibson assembly reaction.

Colony PCR Verification
Following the Gibson assembly, colony PCR was conducted to further verify the construct. The amplification results indicated the presence of the correctly constructed plasmid in multiple positive colonies.

Colony PCR results showing multiple positive colonies containing the correctly constructed pY38a-DpnI.

Enzyme Digestion Verification

We further verified the construct through EcoRI enzyme digestion. The gel electrophoresis results show the expected fragment sizes for pY38a-DpnI (2481 bp and 1845 bp), confirming the correct assembly of the plasmid.

Sequencing Verification

To ensure that the plasmid was correctly constructed, the pY38a-DpnI construct was subjected to sequencing. The sequencing results confirmed that the entire sequence was assembled accurately, with no mutations or unexpected errors in the sequence.

Growth Curve Experiment

To verify the temperature-regulated function of Y38-DpnI-TrrnB, we conducted a growth curve experiment to measure cell growth under different temperature conditions. The results show normal growth at 30°C and 33°C, while at 37°C, the Y38 promoter activates DpnI expression, leading to cell death and a significant decrease in OD600.

Growth curves under different temperature conditions, showing temperature-dependent cell death at 37°C.

Application Extension: Cell-Free System Design

In addition to in vivo applications, we plan to use the Y38-DpnI-TrrnB part to design a cell-free system. At low temperatures (30°C), this system can efficiently induce protein expression. As the temperature increases to 37°C, the Y38 promoter activates DpnI expression, leading to DNA cleavage, termination of protein synthesis, and system shutdown.
This system offers a wide range of applications, including automated process control in synthetic biology, where precise regulation of protein synthesis and DNA degradation is required.

Conclusion

Through the above experiments, we successfully demonstrated the ability of the Y38-DpnI-TrrnB composite part to control cell fate through temperature-dependent regulation. The combination of the Y38 promoter and DpnI provides a highly controllable gene expression system that has potential applications not only in biosafety but also in the development of temperature-controlled cell-free systems.

References

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