Chimeric Plasmid Library Construction via OGAB and Barcode Copying
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Solution Overview
Problem
Constructing a long-chain DNA combinatorial library efficiently is challenging due to difficulties in achieving equal molar concentrations of gene fragments, which hinders the scalability and speed of the DBTL cycle in synthetic biology.
Innovation Solution
The method involves preparing seed plasmids with equimolar unit DNA mixtures using the OGAB method, cleaving them with restriction enzymes to achieve equimolar ratios, and re-assembling these units to construct a long-chain DNA combinatorial library, allowing for efficient microbial cell transformation without confirming the base sequence.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional gene assembling methods are used to construct long-chain DNA combinatorial libraries, then the base sequence can be confirmed through sequencing, but the process is time-consuming and the scaling efficiency is poor
Solution Approach 1:
The patent uses barcode sequences as copies of genotype information that can be rapidly read without sequencing the entire long-chain DNA. Instead of confirming the complete base sequence through time-consuming sequencing, the patent employs barcode copying where each gene fragment carries a unique barcode that represents its identity, allowing fast identification of library contents
Solution Approach 2:
The patent introduces barcode sequences as intermediary elements that mediate between the genotype (gene fragments) and phenotype (library characteristics). These barcodes serve as intermediaries that can be rapidly detected and used to infer the composition of long-chain DNAs without direct sequencing
2Quantity of substance
If all materials are mixed to construct a library with one gene assembling, then a large-scale combinatorial library can be obtained, but the base sequence confirmation becomes the rate-determining step
Solution Approach 1:
The patent replaces time-consuming base sequence sequencing with rapid barcode reading. Each gene fragment in the large-scale library carries a barcode copy of its identity information, allowing the genotype to be confirmed quickly without sequencing the entire long-chain DNA structure
Solution Approach 2:
The patent changes the detection parameter from base sequence composition to barcode sequence composition. By detecting barcode sequences instead of sequencing entire genes, the patent dramatically reduces the time required for genotype confirmation while maintaining library scale
3Manufacturing precision
If gene fragments are prepared individually to achieve equal molar concentrations, then the combinatorial library can be constructed with proper stoichiometry, but the process is complex and difficult to scale
Solution Approach 1:
The patent merges multiple gene fragments with different barcodes into a single equimolar mixture solution. By combining fragments that all share the same overhang sequences and can be assembled into the same structural positions, the patent achieves equal molar concentrations without individually preparing each fragment type
Solution Approach 2:
The patent designs gene fragments with universal overhang sequences that allow them to function in multiple assembly positions. This universality enables different fragments to be mixed in equimolar concentrations since they all can occupy equivalent structural roles in the final long-chain DNA
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 approach significantly enhances the efficiency of constructing long-chain DNA combinatorial libraries, enabling rapid preparation for the next DBTL cycle and allowing reuse of selected plasmids for new libraries, thus overcoming the limitations of existing methods.
Implementation Method 1
processing the plurality of types of plasmids prepared in step (A) with a restriction enzyme suitable for each plasmid to cleave the plasmids into unit DNAs
Data Source
AI summary
The present invention addresses the problem of providing a novel method which is for preparing a DNA fragment for microbial cell transformation, and by which the combinatorial library of a long-chain DNA can be efficiently constructed and confirmation of the genotype of the obtained clone is facilitated. The present invention is a method for preparing a DNA fragment, which is for microbial cell transformation and has at least one insert DNA unit that includes a DNA containing an effective replication origin in a host microorganism and an insert DNA in which unit DNAs are linked, the method being characterized by including: (A) a step for preparing, through an OGAB method, a plurality of types of plasmids having an insert DNA unit in which a plurality of types of unit DNAs capable of being linked in a specific linking order are linked; (B) a step for decomposing a plasmid into unit DNAs by treating the plurality of types of plasmids prepared in the step (A) with a restriction enzyme suitable for each plasmid and preparing a mixed liquid of a plurality of types of unit DNAs; and (C) a step for preparing a long-chain DNA fragment by re-assembling the unit DNAs through the OGAB method by using the mixed liquid of a plurality of types of unit DNAs obtained in the step (B).