Plasmid DNA Non-Standard Nucleotide Conversion in E. coli
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods struggle to introduce and replicate DNA containing non-standard nucleotides in standard cloning systems, leading to issues like mismatching and loss of non-standard nucleotides during PCR reactions, which limits the utility of expanded genetic alphabets in biological systems.
Innovation Solution
The introduction of plasmid DNA with non-standard nucleotide pairs Z:P or S:B into E. coli, where these pairs are converted into standard nucleotides with controlled replacement, allowing for the assembly and replication of DNA constructs using expanded genetic information systems.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If non-standard nucleotide pairs (Z:P or S:B) are introduced into standard cloning systems, then the expanded genetic alphabet can be utilized, but mismatching and loss of non-standard nucleotides occur during PCR reactions
Solution Approach 1:
The patent introduces a conversion system that acts as an intermediary between non-standard nucleotides and standard nucleotides. The plasmid contains both non-standard nucleotide pairs (Z:P or S:B) and standard nucleotide pairs, along with conversion sequences that enable transformation of non-standard pairs into standard pairs. This intermediary conversion mechanism allows the system to first utilize non-standard nucleotides for expanded genetic information storage, then reliably replicate the information by converting to standard nucleotides that are stable in PCR reactions.
Solution Approach 2:
The patent changes the nucleotide composition parameter of the plasmid DNA by incorporating non-standard nucleotide pairs (Z:P or S:B) alongside standard pairs. The plasmid is designed with specific regions containing non-standard nucleotides that can be converted to standard nucleotides through cellular enzymes or chemical treatment, thereby changing the chemical composition from non-standard to standard nucleotides while maintaining the genetic information.
2Loss of information
If non-standard nucleotides are replicated in standard cloning systems, then genetic information can be stored, but loss of non-standard nucleotides occurs during PCR
Solution Approach 1:
The plasmid structure serves as an intermediary that temporarily holds non-standard nucleotide information during assembly, then facilitates its conversion to standard nucleotides for stable replication. The conversion sequences act as mediators that guide the transformation process, ensuring that genetic information is preserved while the physical nucleotide composition changes from non-standard to standard forms suitable for PCR.
Solution Approach 2:
The patent performs preliminary assembly of the plasmid containing non-standard nucleotides in a controlled environment where they are stable, before introducing the plasmid into bacterial cells for replication. This preliminary action allows the non-standard nucleotides to be properly incorporated and positioned in the plasmid structure before the conversion to standard nucleotides occurs during bacterial replication or subsequent PCR steps.
3Productivity
If standard cloning systems are used, then PCR reactions can proceed, but mismatching occurs with non-standard nucleotide pairs
Solution Approach 1:
The conversion system acts as an intermediary step between plasmid assembly and PCR amplification. The plasmid is first assembled with non-standard nucleotides in a controlled setting, then introduced into bacterial cells where conversion mechanisms transform non-standard pairs into standard pairs. This intermediary conversion ensures that by the time PCR is performed, only standard nucleotide pairs remain, eliminating mismatching while preserving the productivity benefits of the expanded genetic alphabet during the assembly phase.
Solution Approach 2:
The patent employs a periodic or sequential process where non-standard nucleotides are first utilized for information encoding during plasmid construction, then converted to standard nucleotides before PCR amplification. This periodic switching between using non-standard and standard nucleotides at different stages of the workflow allows the system to benefit from both expanded genetic capacity and reliable PCR replication without simultaneous conflicts.
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 enables the stable conversion of non-standard nucleotides into standard ones within E. coli, facilitating the assembly and replication of DNA constructs while maintaining the orthogonality and specificity of non-standard pairs, thus overcoming the limitations of mismatching and loss in standard cloning systems.
Implementation Method 1
when introduced as a vector into E. coli, leads to a plasmid where the non-standard nucleotides are not lost by deletion, but rather are converted into standard nucleotides
Data Source
AI summary
The instant invention provides for the assembly of large DNA oligonucleotide constructs by the self-assembly of multiple oligonucleotide fragments, wherein the assembly is guided by the hybridization between non-standard nucleotides that form non-standard nucleobase pairs orthogonal to the standard T:A and C:G nucleobase pairs. Adding nucleobase pairs increases the information density of the fragments, minimizing off-target hybridization. The invention further provides rules and methods for converting non-standard pairs into standard pairs using polymerase copying with conversion.