Programmable Artificial Cell Compartmentalization
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current artificial cell systems face challenges in performing protein synthesis and post-translational modifications (PTMs) in a controlled and simple manner, lacking the ability to spatially and temporally segregate central dogma activities, which complicates protein production and makes it difficult to profile and characterize PTMs effectively.
Innovation Solution
A cell-free system comprising hierarchical compartments for transcription, translation, and PTMs, allowing for the processing and transport of products through connecting channels, enabling precise control over protein production and modification, including the use of microbeads and specific binding pairs to manage RNA polymerase, DNA, and proteins within the system.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If artificial cell systems use elaborate procedures with multiple steps to perform protein synthesis, then protein production capability is achieved, but device complexity and operational difficulty increase
Solution Approach 1:
The system divides the artificial cell into distinct hierarchical compartments: a transcription compartment containing DNA and RNA polymerase, a transport compartment with size-selective barriers, and a translation compartment with ribosomes and translation factors. This segmentation allows each compartment to perform its specific function independently while maintaining overall system productivity, resolving the contradiction between complex multi-step procedures and simplified operation.
2Manufacturing precision
If artificial cell systems lack spatial and temporal segregation of central dogma activities, then device simplicity is maintained, but manufacturing precision and control over protein production deteriorate
Solution Approach 1:
The invention implements spatial segregation by placing transcription machinery (DNA, RNA polymerase) in a transcription compartment and translation machinery (ribosomes, translation factors) in a separate translation compartment. Size-selective barrier membranes control the temporal sequence of events by allowing RNA to pass from transcription to translation while retaining DNA and ribosomes in their respective compartments. This segmentation achieves precise control over protein production without requiring elaborate external procedures.
Solution Approach 2:
The size-selective barrier membranes act as intermediaries that selectively permit RNA to pass from the transcription compartment to the translation compartment while blocking DNA and larger macromolecular complexes. This intermediary mechanism enables automatic spatial and temporal control of central dogma activities, achieving manufacturing precision without complex external intervention.
3Adaptability or versatility
If PTMs are synthetically introduced in an indiscriminate manner, then protein modification capability is achieved, but measurement precision and characterization difficulty increase
Solution Approach 1:
The invention introduces site-specific post-translational modifications by incorporating modified amino acids (e.g., phosphoamino acids, glycosylated amino acids) into the translation compartment. These modifications occur locally at specific positions determined by the mRNA sequence and tRNA availability, rather than indiscriminately throughout the protein. This local quality approach enables precise characterization of PTMs while maintaining versatility in modification types.
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 allows for the production of purified, modified proteins in a single device, enabling precise control over PTMs and simplifying the production of complex biomolecules, as demonstrated by the synthesis of ubiquitinated α-synuclein, which can be applied to various protein-based systems.
Implementation Method 1
an inner surface of the transcription compartment 101 comprises a first functional group of a specific binding pair capable of binding to a complementary second functional group of the binding pair present on the RNA polymerase
Implementation Method 2
a barrier for selectively preventing or substantially decreasing passage of DNA or modified-DNA therethrough, wherein the barrier is designed to bind a molecule capable of selectively binding DNA or modified-DNA
Implementation Method 3
retain microbeads capable of binding an RNA polymerase
Implementation Method 4
the compartment 102 is designed to bind the protein or retain microbeads capable of binding the protein
Data Source
AI summary
The present invention provides cell-free systems comprising: (i) a transcription compartment; (ii) a barrier; and (iii) a translation compartment, methods for producing proteins and performing post-translational modifications thereon using such systems, and kits comprising such systems.