Loopable mRNA Translation for Stable Repetitive Protein Synthesis
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Solution Overview
Problem
Existing technologies face challenges in synthesizing large repetitive proteins due to genetic instability, inconvenient manipulation of large plasmids, and high metabolic costs, making it difficult to recreate materials like spider silks and squid ring teeth proteins.
Innovation Solution
A genetic construct using a permuted Group I self-splicing intron to circularize mRNA, allowing ribosomes to translate repetitive protein sequences efficiently without large DNA constructs, incorporating a TEV protease cleavage site and optimized ribosome binding sites for enhanced translation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Length of moving object
If rolling circle amplification is used to generate large repetitive proteins, then protein size can be increased, but genetic instability increases due to spontaneous recombination
Solution Approach 1:
The genetic construct is divided into a small core repetitive unit (less than 100 amino acids) flanked by unique sequences. The repetitive protein is generated by translational recycling rather than DNA amplification, segmenting the function between a stable small DNA template and a large protein product.
Solution Approach 2:
A Group I self-splicing intron acts as an intermediary element that enables mRNA circularization and ribosome recycling. The intron's splicing activity creates the conditions for translational looping without requiring large repetitive DNA sequences, mediating between the small DNA template and large protein output.
2Productivity
If large plasmids containing tandem repeat proteins are used, then protein expression capacity increases, but ease of manipulation decreases for PCR, sequencing, and transformation
Solution Approach 1:
The system segments the genetic information into a compact plasmid containing only the essential repetitive unit and flanking sequences, separating the template storage function from the protein synthesis function. This allows standard molecular biology techniques to be applied to the small plasmid while still producing large proteins.
Solution Approach 2:
The protein sequence is copied repeatedly during translation through ribosome recycling on circularized mRNA, rather than copying the DNA template repeatedly. This allows protein amplification without plasmid amplification, maintaining ease of DNA manipulation.
3Productivity
If large DNA and mRNA molecules are replicated and transcribed, then protein production capacity increases, but metabolic cost increases
Solution Approach 1:
The ribosome continuously recycles on the circularized mRNA, performing repeated translation cycles without requiring new mRNA transcription for each protein copy. This continuous translational action on a stable mRNA template reduces the metabolic cost of repeated gene expression.
Solution Approach 2:
The system copies the protein product through translational recycling rather than copying the genetic template repeatedly. A single small DNA template and its corresponding mRNA are used to generate multiple protein copies, reducing the metabolic burden of template replication and transcription.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The construct enables stable production of repetitive proteins with high efficiency, reducing genetic instability and metabolic costs, and allows for the creation of materials like dragline silk and biofilms with hierarchical structures.
Implementation Method 1
a 3′ portion of a Group I self-splicing intron
Implementation Method 2
auto-catalytically inducing RNA sequences to form a closed loop
Implementation Method 3
enabling the creation of repetitive protein sequences
Data Source
AI summary
The present disclosure is directed to a genetic construct for performing loopable translation, a kit comprising the genetic construct, and a method of producing biomaterials comprising a highly repetitive protein by use of the genetic construct.