Protein Ionic Liquid Transfection for Microorganisms
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Solution Overview
Problem
Current methods for transfecting microorganisms with proteins are limited, as they often result in permanent genetic modifications and lack universal protein transfection reagents for gram-negative bacteria, and existing protein/DNA transfection reagents for mammalian cells are expensive and toxic.
Innovation Solution
The use of protein ionic liquids for intracellular delivery and transfection of microorganisms, which allows for temporary modification of cells without permanent genetic changes, using methods such as inoculating microorganisms in growth media, growing and harvesting cells, adding protein ionic liquids, freezing, and removing water to create a cell powder for reconstitution and transfection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If DNA plasmids are used for transformation, then microorganisms can perform recombinant protein expression, but permanent genetic modifications are introduced that are passed to all daughter cells
Solution Approach 1:
The patent uses protein ionic liquids as temporary, non-integrating transfection agents that deliver functional proteins without permanent genetic modification. The proteins are introduced transiently and are not inherited by daughter cells, allowing recombinant protein expression without stable genetic transformation.
2Ease of operation
If protein/DNA transfection reagents for mammalian cells are used, then protein transfection can be achieved, but the reagents are expensive and toxic
Solution Approach 1:
The patent employs protein ionic liquids as inexpensive, non-toxic alternatives to commercial transfection reagents. These reagents are based on biocompatible ionic liquid chemistry that is far less toxic and significantly cheaper than lipid-based or viral transfection systems used for mammalian cells.
Solution Approach 2:
The patent uses composite protein ionic liquid structures that combine cationic proteins with anionic ionic liquid components. This composite approach creates effective transfection agents that are both biocompatible and functional, avoiding the toxicity of pure cationic lipids or complex viral vectors.
3Adaptability or versatility
If universal protein transfection reagents are developed, then transfection of gram-negative bacteria can be achieved, but no such reagents currently exist
Solution Approach 1:
The patent develops protein ionic liquids that function as universal transfection agents for gram-negative bacteria, yeast, and other microorganisms. The ionic liquid chemistry provides a platform that can deliver various proteins across different microbial species without requiring species-specific optimization, achieving broad universality.
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 temporary introduction of functional proteins into microorganisms, providing a non-genetic method for modifying cells, eliminating the risk of GMO introduction and offering a universal, inexpensive transfection agent for microorganisms, suitable for applications like synthetic biology and living inks.
Implementation Method 1
The disclosed use of protein ionic liquids represents the first non-genetic approach for modifying cells with proteins that lack permanent genetic modifications
Implementation Method 2
freezing the suspended cells between -20 to -212°C
Implementation Method 3
removing at least 99% of water from the frozen suspended cells to make a cell powder
Data Source
AI summary
A method for transfecting microorganisms comprises inoculating a growth media consisting of at least one of sterile LB media and tryptic soy broth with microorganism cells (cells) consisting of at least one of E.coli (DH5α), C. lytica, or B. subtilus, Pichia pastoris; growing the cells at between 28-40° C. to achieve a desired cell density; harvesting the cells; adding a protein ionic liquid consisting of at least one of green fluorescent protein (GFP), ferritin, rabbit IgG antibodies, and photosystem II from spinach ionic liquid to the cells; suspending the cells in the protein ionic liquid; freezing the suspended cells between −20 to −212° C.; and removing at least 99% of water from the frozen suspended cells to make a cell powder. The cell powder may be reconstituted in Tris HCl buffer and mixed to obtain uniform cell suspension; and centrifuged to obtain cell pellet.