TNT Cloning System Using Type IIS Enzymes for Sequence-Independent Assembly
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Solution Overview
Problem
Current cloning methods face limitations in flexibility, fidelity, and universality, particularly in handling multiple DNA segments, due to requirements for sequence overlap, error-prone PCR, and specific recognition sequences, which restrict the assembly of genetic elements and regulatory regions in synthetic biology and genetic circuit engineering.
Innovation Solution
A cloning system utilizing two families of double-stranded vectors with shared Type IIS restriction enzyme sites and pre-selected 3-nucleotide signature elements allows for infinite rounds of cloning, enabling the combination of multiple genetic elements into a final vector without the need for sequence overlap or mutation, using a 'cloning loop' mechanism that automatically matches protein-encoding sequences in frame.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If homology-based cloning methods are used, then cloning can be performed, but sequence overlap is required which limits the type and order of fragment cloning
Solution Approach 1:
The invention extracts and removes the requirement for sequence overlap from the cloning process by using Type IIS restriction enzymes that cut outside their recognition sites, allowing fragments to be joined without homologous sequences
Solution Approach 2:
The invention creates a universal cloning system where the same Type IIS restriction enzymes and signature sequences can be used for any DNA fragment assembly, making the method applicable to all fragments regardless of their sequence content
2Productivity
If PCR-based methods are used, then DNA amplification can be performed, but the methods are error prone
Solution Approach 1:
The invention replaces the error-prone PCR amplification step with a restriction enzyme-based ligation system that directly assembles fragments without requiring thermal cycling and polymerase activity, thereby eliminating PCR-induced errors
3Adaptability or versatility
If restriction enzyme-based methods are used, then cloning can be performed, but specific recognition sequences must be present at specific sites which limits the number of fragments
Solution Approach 1:
The invention introduces signature sequences as intermediaries that are recognized by Type IIS restriction enzymes, allowing any DNA fragment to be cloned by adding these universal signature sequences rather than requiring specific recognition sites within the fragments themselves
4Adaptability or versatility
If GoldenBraid method is used, then endless assembly can be achieved, but multiple libraries and linkers/adaptors are required which complicates the system
Solution Approach 1:
The invention achieves endless assembly using a single universal library system where all fragments use the same Type IIS restriction enzyme sites and signature sequences, eliminating the need for multiple specialized libraries and linkers required by GoldenBraid
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 system facilitates the efficient and flexible assembly of multiple genetic elements, eliminating the use of linkers and adaptors, and supports the creation of complex constructs by allowing continuous cloning loops, thereby overcoming the limitations of existing methods.
Implementation Method 1
utilizing two families of double-stranded vectors with shared Type IIS restriction enzyme sites
Implementation Method 2
allowing continuous cloning loops, thereby overcoming the limitations of existing methods
Data Source
AI summary
Disclosed herein are vectors and components for a nucleic acid cloning system, and methods of use of the vectors and components in cloning nucleic acid fragments of interest. The cloning system includes two families of destination vectors which can be used in alternating form to systematically combine nucleic acid fragments of interest.