Recombinase-Based DNA State Machines for Temporal Event Recording
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Solution Overview
Problem
Current methods lack a scalable and generalizable framework for implementing complex functional state machines in living cells, which are essential for understanding and engineering biological systems, as they require stable storage of state information with minimal cellular burden.
Innovation Solution
A recombinase-based framework that encodes state in DNA sequences using chemically controlled DNA excision and inversion operations, allowing for the implementation of state machines in living cells that can record the identities and orders of gene regulatory events and control gene expression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If recombinase-based state machines are implemented in living cells, then the ability to record temporal order and perform complex gene regulation is improved, but the device complexity and cellular burden increase
Solution Approach 1:
The system segments the state machine functionality into discrete modular components: multiple serine recombinases (Bxb1, Tp901, A118, PhIF, AraC) each recognizing specific RRS pairs, with states encoded by distinct DNA sequence arrangements. This modular segmentation allows complex temporal logic to be built from simple, interchangeable units that can be independently characterized and combined.
Solution Approach 2:
The patent implements nesting by arranging RRS pairs in overlapping configurations where recombination events are nested within each other. The DNA sequence contains multiple nested RRS pairs that are processed in a specific temporal order, with each recombination event modifying the DNA structure to enable or disable subsequent recombination events, creating a nested hierarchy of regulatory control.
2Quantity of substance
If multiple serine recombinases with overlapping RRS pairs are used, then the number of recordable states increases, but the manufacturing precision and design difficulty increase
Solution Approach 1:
The patent employs universal design principles where standardized RRS pairs (attB/attP variants) can be combined in different configurations to create multiple distinct states. The same basic RRS pair design principles apply across all five serine recombinases, allowing the system to scale from 2-input to 3-input configurations without requiring fundamentally new components, only different arrangements of the universal building blocks.
Solution Approach 2:
The patent uses preliminary action by pre-designing and pre-characterizing the recombination outcomes for each RRS pair configuration. The DNA sequence is engineered in advance with specific RRS pair arrangements that are predetermined to produce the desired state transitions. The overlapping configuration is pre-planned so that recombination events automatically occur in the correct temporal sequence without requiring real-time control.
3Measurement precision
If chemically controlled DNA excision and inversion operations are used, then the control precision over gene expression is improved, but the measurement and detection difficulty increases
Solution Approach 1:
The patent employs fluorescent reporter genes (GFP, RFP, BFP) as visual indicators of state machine output. Each gene expression state produces a distinct fluorescent signal that can be easily detected by flow cytometry or fluorescence microscopy. This converts the invisible DNA recombination events into visible optical signals, making precise control measurements straightforward while maintaining high detection sensitivity.
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
Enables the creation of scalable state machines in living cells that can record temporal and combinational events, perform multi-input, multi-output control of gene expression, and facilitate the programming of sophisticated cell behaviors, improving our understanding of biological processes and potential therapeutic applications.
Implementation Method 1
recombinase-based framework for building state machines in living cells by using chemically controlled DNA excision and inversion operations to encode state in DNA sequence
Data Source
AI summary
Provided herein are recombinase-based frameworks for building state machines in vitro and in vivo by using chemically controlled DNA excision and inversion operations to encode state in DNA sequence.