A method for quickly constructing a single-plasmid T7 expression system

By predicting and adjusting the lethal upper limit of T7 RNAP in the single-plasmid T7 expression system, the problems of complexity and long construction time in the application of T7 expression system in non-model hosts are solved, realizing rapid and customized construction of single-plasmid T7 expression system, reducing host mortality risk and improving construction success rate.

CN119144632BActive Publication Date: 2026-06-23TSINGHUA UNIVERSITY
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Patent Information

Application Number
CN202310685766.7
Authority / Receiving Office
CN · China
Patent Type
Patents(China)
Current Assignee / Owner
Filing Date
2023-06-09
Publication Date
2026-06-23
Estimated Expiration
2043-06-09

AI Technical Summary

Technical Problem

In existing technologies, the application of T7 expression systems in non-model hosts is complex. The construction of single-plasmid T7 expression systems is time-consuming and has a low success rate. Furthermore, host bacteria are prone to death due to overexpression of T7 RNAP.

Method used

This paper presents a method for rapidly constructing a single-plasmid T7 expression system based on the modular concept of synthetic biology. By predicting the lethal upper limit of T7 RNAP in the host, the DNA structure of the T7 RNAP expression unit is adjusted, including changing the promoter, RBS, codon, terminator, plasmid copy number, etc., and inserting antisense RNA and transcription interference structures to achieve host-specific and customized construction.

Benefits of technology

This method enables the rapid construction of single-plasmid T7 expression systems in different hosts, reduces T7 RNAP expression levels and activity, improves construction success rate, simplifies the operation process, and provides a convenient method for applying T7 expression systems to non-model strains.

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Abstract

The application provides a method for constructing a T7RNAP expression unit, which comprises the following steps: inserting a designed T7RNAP expression unit into a test plasmid to express T7RNAP in a host cell, then determining the T7RNAP enzyme activity E expressed by the plasmid, if E < E0, the threshold value of the T7RNAP enzyme activity in the host cell, then the T7RNAP expression unit is successfully constructed, if E ≥ E0, the DNA structure of the designed T7RNAP expression unit is adjusted, and the above steps are repeated until E < E0, then the T7RNAP expression unit is successfully constructed, and finally the plasmid with the T7RNAP enzyme activity lower than E0 is selected. The application also provides a method for adjusting the DNA structure of the designed T7RNAP expression unit in the above steps. The method for constructing the T7RNAP expression unit has the characteristics of predictability, rapidity, customization and the like, thereby solving the problems of the previous single-plasmid T7 expression system, such as incapability of construction, long construction period and easy mutation of the system, and providing a convenient method for the non-model strains to use the T7 expression system.
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