Aspergillus niger Spore Protein Expression via HDEN Electroporation
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods fail to directly introduce exogenous single-stranded RNA into dormant spores of Aspergillus niger, due to low cell permeability and high RNAse activity, which hinders the expression of proteins in these spores without the need for germination.
Innovation Solution
Employing the HDEN electro-transformation technique to deliver exogenous single-stranded RNA directly into the dormant spores of Aspergillus niger, bypassing the germination step and utilizing specific electroporation buffers and conditions to enhance RNA uptake and protein expression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional methods are used to introduce exogenous RNA into Aspergillus niger spores, then the process requires spore germination and complex DNA plasmid construction, but this increases process complexity and time consumption
Solution Approach 1:
The invention extracts and utilizes only the essential component (exogenous single-stranded RNA) needed for protein expression, eliminating the need for complex DNA plasmid construction and spore germination steps. The RNA is directly introduced into dormant spores through electroporation, achieving protein expression without the cumbersome DNA-based workflow.
Solution Approach 2:
The exogenous single-stranded RNA is prepared in advance with appropriate structural modifications (such as 5' cap and poly-A tail) to ensure stability and translation efficiency. This preliminary preparation of the RNA molecule allows it to function directly upon introduction into the spores, eliminating the need for in vivo transcription from DNA plasmids.
2Reliability
If exogenous single-stranded RNA is introduced into dormant spores, then protein expression can be achieved without germination, but low cell permeability and high RNAse activity prevent RNA uptake
Solution Approach 1:
The invention employs periodic electroporation pulses to temporarily create pores in the spore cell wall and membrane, allowing exogenous RNA to enter. The electroporation process uses controlled electrical pulses that open channels for RNA uptake, then allow the membrane to reseal, protecting the introduced RNA from degradation by RNAse while maintaining spore dormancy.
Solution Approach 2:
The invention changes the physical state of the spore membrane through electroporation (applying electrical field parameters), temporarily increasing permeability to allow RNA entry. Additionally, the RNA is chemically modified with protective structures (5' cap, poly-A tail) that change its resistance to degradation, enabling it to withstand the high RNAse activity in dormant spores.
3Duration of action of stationary object
If DNA plasmids are used for protein expression, then stable inheritance and continuous protein production are achieved, but this requires spore germination and affects the haploid status
Solution Approach 1:
The invention uses a disposable exogenous RNA molecule that performs its function (protein expression) within a limited timeframe without requiring stable inheritance. The RNA is introduced into dormant spores, expresses protein immediately, and is then degraded naturally, eliminating the need for long-term stable integration into the genome or continuous replication that would require germination.
Data Source
AI summary
A method to produce protein in Aspergillus niger's sleeping spores using single-stranded RNA is provided. The method includes three steps: culture of Aspergillus niger and collection of spores, pretreatment of Aspergillus niger spores, and electroporation of Aspergillus niger spores using HDEN method. Non-germinated spores are used as a starting material for introduction of exogenous molecules. The exogenous protein coding single-stranded RNA is introduced into the resting spores of Aspergillus niger by employing the HDEN electrotransformation technique to express protein. This method is simple and fast, the effect is excellent, and the transformation rate reaches more than 90%.