Eukaryotic Pol-2 Promoter Expression in Prokaryotes
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Solution Overview
Problem
Current methods for producing eukaryotic RNAs and proteins in prokaryotic cells are limited by the incompatibility between prokaryotic and eukaryotic transcription systems, leading to error-prone processes and the need for costly and laborious hybridoma or mammalian cell cultures, as well as the use of antibiotics that can inhibit prokaryotic growth.
Innovation Solution
A composition using chemical agents such as 3-morpholinopropane-1-sulfonic acid (MOPS), ethanol, or glycerin to induce eukaryotic pol-2 promoter-driven transcription in prokaryotes, allowing for the direct expression of desired RNAs and proteins without requiring a change to error-prone prokaryotic promoters or the use of expensive cell cultures, and enabling improved reading fidelity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If bacterial or bacteriophage promoters are used in prokaryotic cells for gene expression, then the transcription process can proceed, but the process becomes error-prone and mutations occur
Solution Approach 1:
The patent changes the promoter type parameter from traditional bacterial promoters (T7, T3, SP6, Lac, Tac) to eukaryotic RNA polymerase II promoters. This parameter change fundamentally alters the transcription mechanism to use eukaryotic transcription factors and machinery, which inherently provides higher fidelity and reduces mutation rates during transcription in prokaryotic cells.
2Manufacturing precision
If eukaryotic RNA polymerase II promoter-driven transcription is attempted in prokaryotes, then reading fidelity improves, but the transcription system incompatibility prevents direct expression
Solution Approach 1:
The patent introduces eukaryotic transcription factors as intermediary molecules that bridge the incompatibility between prokaryotic transcription machinery and eukaryotic RNA polymerase II promoters. These transcription factors bind to the eukaryotic promoter sequences and recruit the RNA polymerase II complex, enabling functional coupling between prokaryotic cellular environment and eukaryotic promoter elements.
Solution Approach 2:
The patent creates a universal expression system where eukaryotic RNA polymerase II promoters can function in both eukaryotic and prokaryotic cells. By combining eukaryotic promoters with prokaryotic transcription factors and RNA polymerase, the system achieves multi-functionality across different cellular domains, allowing the same promoter architecture to drive gene expression universally.
3Ease of operation
If traditional inducible gene expression methods using antibiotics are employed, then gene expression can be controlled, but prokaryotic growth is inhibited
Solution Approach 1:
The patent extracts and eliminates the harmful antibiotic component from traditional inducible expression systems. Instead of using antibiotic-based induction (which inhibits prokaryotic growth), the invention employs alternative induction mechanisms such as chemical inducers or physiological triggers that do not interfere with bacterial metabolism and growth, thereby separating the gene expression control function from the growth-inhibiting effect.
4Productivity
If prokaryotic cells are used for producing eukaryotic RNAs and proteins, then cost-effective bulk production is achieved, but the incompatible transcription systems prevent direct expression from eukaryotic promoters
Solution Approach 1:
The patent segments the transcription system into modular components: eukaryotic promoter elements, prokaryotic RNA polymerase, and prokaryotic transcription factors. This segmentation allows each component to be optimized for its native environment while working together in a hybrid system, enabling prokaryotic cells to express eukaryotic genes with high efficiency and fidelity.
Data Source
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AI summary
Eukaryotic protein-coding messenger RNAs and non-coding microRNAs are naturally transcribed by type II RNA polymerases (pol-2) but not prokaryotic RNA polymerases. As a result, current eukaryotic RNA and protein production is performed either using eukaryotic pol-2 promoters in hybridomas or mammalian cells or using prokaryotic promoters in bacterial cells. However, because prokaryotic RNA transcription tends to be error-prone, frequent mutation is a big problem. Also, growing hybridomas or mammalian cells is relatively laborious and costly. To overcome these problems, the present invention provides a novel inducible composition and method for producing eukaryotic RNAs and/or their related peptides/proteins directly using eukaryotic pol-2 promoter-driven gene expression in fast growing bacteria, without the need of changing to prokaryotic promoters or growing hybridomas/mammalian cells. The RNAs and peptides/proteins so obtained can be used to develop drugs, cure diseases, treat tumors/cancers, produce pluripotent stem (iPS) cells, enhance wound healing, and make foods.