Mtb ATP Synthase Expression in Mycobacterium smegmatis
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
The development of novel anti-tuberculosis drugs is hindered by the difficulty in expressing and purifying Mycobacterium tuberculosis (Mtb) ATP synthase, due to its differences from Mycobacterium smegmatis ATP synthase, and the unclear mechanism of action of small-molecule inhibitors, which restricts the precise assembly and functional understanding of Mtb ATP synthase.
Innovation Solution
A construction method for a recombinant bacterium that expresses Mtb ATP synthase by transferring the Mtb ATP synthase gene cluster into a Mycobacterium smegmatis competent cell, knocking out the native ATP synthase genome, and using a prokaryotic expression vector to achieve induced expression and purification of Mtb ATP synthase, thereby avoiding biological hazards and hybrid expression issues.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If Mtb ATP synthase is expressed in Msm through heterologous expression, then Mtb ATP synthase can be produced, but the native Msm ATP synthase gene must be knocked out which is difficult to achieve
Solution Approach 1:
The patent applies preliminary action by first introducing the Mtb ATP synthase gene cluster into the Msm competent cell before knocking out the native Msm ATP synthase gene. This ensures that the Mtb ATP synthase is already present and functional before the knockout occurs, avoiding the problem of losing the native gene during the expression process. The auxiliary gene knockout plasmid is introduced in advance to facilitate the subsequent knockout step.
Solution Approach 2:
The patent uses an auxiliary gene knockout plasmid as an intermediary tool to achieve the gene knockout. This plasmid contains the Msm ATP synthase gene flanked by homologous sequences and a selectable marker, enabling precise knockout of the native Msm ATP synthase gene through homologous recombination. The plasmid acts as a mediator between the gene knockout objective and the cellular machinery.
2Productivity
If Mtb ATP synthase is expressed in Msm, then ATP synthase can be produced, but hybrid complex formation occurs between Mtb and Msm ATP synthase components
Solution Approach 1:
The patent applies the extraction principle by completely removing the native Msm ATP synthase gene from the host cell genome through knockout. This ensures that only the Mtb ATP synthase components are present in the cell, eliminating the possibility of hybrid complex formation between Mtb and Msm ATP synthase proteins. The extraction of the native gene is achieved through precise homologous recombination mediated by the auxiliary knockout plasmid.
Solution Approach 2:
The patent applies local quality by introducing the Mtb ATP synthase gene cluster with an affinity purification tag at a specific locus (sodC gene locus) in the Msm genome. This localized insertion ensures that the Mtb ATP synthase is expressed in a controlled manner with specific properties (affinity tag for purification) while the rest of the cell maintains its normal Msm characteristics, preventing widespread hybridization.
3Productivity
If Mtb ATP synthase is expressed, then protein can be produced for study, but the process is complex and costly
Solution Approach 1:
The patent applies universality by using Msm as a host system that can perform multiple functions: it provides the cellular machinery for protein expression, serves as a model organism with well-characterized genetics for precise gene manipulation, and offers a safe alternative to Mtb for heterologous expression. The Msm host combines the benefits of ease of manipulation with the ability to express Mtb proteins, creating a multi-functional platform.
Solution Approach 2:
The patent applies copying by creating a simplified version of the Mtb ATP synthase expression system in Msm. Instead of working with the complex Mtb system directly, the patent copies the essential functional elements (ATP synthase gene cluster) into the simpler Msm host, enabling protein production with reduced complexity. The affinity purification tag serves as a copy of the purification function, simplifying the downstream processing.
Data Source
AI summary
The present disclosure provides a Mycobacterium tuberculosis (Mtb) adenosine triphosphate (ATP) synthase-expressing recombinant bacterium, and a construction method and an expression method thereof, and belongs to the technical field of exogenous expression. The present disclosure provides a construction method of an Mtb ATP synthase-expressing recombinant bacterium, including the following steps: using a Mycobacterium smegmatis (Msm) competent cell containing an auxiliary gene knockout plasmid as a basal cell; transferring an Mtb ATP synthase gene cluster with an affinity purification tag into the basal cell, knocking out a Msm ATP synthase genome, and collecting a strain without the auxiliary gene knockout plasmid after conducting repeated subculture; and transferring the Mtb ATP synthase gene cluster into the strain by prokaryotic expression to obtain the Mtb ATP synthase-expressing recombinant bacterium.