In Vitro SCRaMbLE System for DNA Library Construction

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Solution Overview

Problem

The complexity of biological systems and the number of genes in synthetic DNA designs pose challenges in ensuring that synthetic DNA carries out intended functions, particularly in debugging and optimizing biosynthetic pathways, where existing methods lack flexibility and efficiency in in vitro assembly and optimization.

Innovation Solution

The in vitro SCRaMbLE system uses recombinant Cre recombinase mixed with purified DNA encoding loxPsym sites to enable top-down and bottom-up approaches for DNA library construction, allowing for rearrangement of transcription units and optimization of biosynthetic pathways through site-specific recombination, thereby correlating phenotype and genotype.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Adaptability or versatility

If traditional in vivo SCRaMbLE system is used, then stochastic diversity in chromosome structure can be generated, but the system lacks flexibility for biochemical optimization and in vitro assembly

Engineering Contradiction:

Improveflexibility for biochemical optimizationVSAvoidsystem complexity

Core Design Contradiction:

Adaptability or versatilityVSDevice complexity

Solution Approach 1:

The patent introduces an intermediary in vitro recombination system that uses purified Cre recombinase enzyme and loxPsym sites as mediators to enable controlled DNA rearrangement without requiring living cells. This intermediary system bridges the gap between traditional in vivo methods and biochemical optimization needs, allowing researchers to perform SCRaMbLE reactions in a controlled test tube environment while maintaining the core recombination mechanism.

Inventive Principle:

Principle #24Intermediary (Mediator)

Solution Approach 2:

The patent replaces the biological mechanical system (in vivo cellular machinery) with a biochemical system using purified enzymes and DNA substrates. By substituting the complex in vivo cellular environment with an in vitro biochemical reaction system, the method enables precise control over recombination conditions while eliminating the need for living cells, thus achieving biochemical optimization flexibility.

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

2Productivity

If in vitro SCRaMbLE system is implemented, then flexibility and efficiency of pathway engineering is enhanced, but the complexity of correlating phenotype and genotype increases

Engineering Contradiction:

Improveefficiency of pathway engineeringVSAvoidcomplexity of correlating phenotype and genotype

Core Design Contradiction:

ProductivityVSDevice complexity

Solution Approach 1:

The patent applies preliminary action by performing in vitro SCRaMbLE reactions to generate diverse DNA libraries before introducing them into host cells for phenotype testing. This allows the genetic diversity to be pre-established and controlled in a biochemical reaction, enabling more efficient screening and correlation of genotypes with phenotypes by starting with a defined library rather than relying on in vivo recombination events.

Inventive Principle:

Principle #10Preliminary action

3Adaptability or versatility

If multiple loxPsym sites are introduced in DNA, then rearrangement diversity is increased, but the difficulty of debugging and ensuring intended function increases

Engineering Contradiction:

Improverearrangement diversityVSAvoiddifficulty of debugging

Core Design Contradiction:

Adaptability or versatilityVSDifficulty of detecting and measuring

Solution Approach 1:

The patent uses the in vitro recombination system as an intermediary to systematically generate and control rearrangements among multiple loxPsym sites. By performing reactions in a controlled biochemical environment with purified enzymes, the system can methodically explore different rearrangement outcomes and facilitate debugging by allowing direct manipulation and analysis of DNA products without the complexity of in vivo cellular processes.

Inventive Principle:

Principle #24Intermediary (Mediator)

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

This method generates diverse DNA libraries that can be biochemically optimized, enhancing the flexibility and efficiency of pathway engineering by modifying gene copy number, order, and orientation, leading to improved biosynthetic pathway flux and product yield, as demonstrated with the β-carotene pathway in yeast.

Implementation Method 1

Cre recombinase catalyzes a site-specific recombination reaction between two loxP sites

Methodology Applied

Scientific EffectSite-specific recombination: Enzyme

Data Source

PatentUS11624069B2In vitro DNA SCRaMbLE

Publication Date: 2023.04.11 NEW YORK UNIV

AI summary

Provided is an in vitro method for making a recombinant DNA molecule. The method includes combining in vitro a recombination-site-mediated evolution (a SCRaMbLE) ready DNA polynucleotide that contains at least one transcription unit (TU) and an introduced site-specific recombinase recognition sites that can be recognized by a recombinase, with a recombinase that recognizes the site-specific recombinase recognition sites. The method results in a polynucleotide that is recombined to provide a recombined polynucleotide. The method may further include determining the sequence, or determining the expression of the recombined polynucleotide. Polynucleotides made by this process, and modified yeast that contain the modified polynucleotides, are also provided.