Intracellular non-genetic modification of microorganisms using protein ionic liquids
Inventors
Slocik, Joseph M • Naik, Rajesh R. • Dennis, Patrick B
Assignees
United States Department of the Air Force
Publication Number
US-11781106-B1
Publication Date
2023-10-10
Expiration Date
2041-03-01
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Abstract
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.
Core Innovation
The invention provides a method for the non-genetic intracellular delivery, loading, and transfection of microorganisms and/or bacterial spores with functional proteins using protein ionic liquids. This method uses water-free protein ionic liquids composed of cationized proteins complexed with anionic polymers to form charge-neutral protein salts that melt near room temperature to highly viscous liquids. The process involves suspending microbial cells with protein ionic liquids, freezing, lyophilizing to remove at least 99% water, and optionally reconstituting the cell powder for further use. This approach enables temporary modification of cell phenotype and proteome without introducing permanent genetic modifications.
The problem addressed is the lack of a non-genetic method to introduce heterologous proteins into microorganisms. Existing transformation methods rely on DNA plasmids leading to permanent genetic changes passed through cell generations, posing environmental risks associated with GMOs. Protein transfection methods common in mammalian cells are expensive, specialized, and toxic, relying on natural endocytosis and specific sequences absent in many microorganisms. There is no universal protein transfection reagent for gram-negative bacteria or yeast, and intracellular protein delivery inside microbial cells has not been a standard practice.
The invention overcomes these limitations by utilizing protein ionic liquids as universal, inexpensive, and general transfection agents that can intracellularly load a variety of proteins (e.g., GFP, ferritin, IgG antibodies, photosystem II) into genetically tractable and intractable microorganisms including E. coli, C. lytica, B. subtilis, and Pichia pastoris. The method allows temporary cell modifications for synthetic biology, cellular ruggedization, or living inks with minimized viability loss. The approach avoids permanent genetic changes, thus eliminating risks of GMO release, and can deliver multiple proteins simultaneously in a single step.
Claims Coverage
The patent includes one independent claim followed by dependent claims that elaborate on specific steps and conditions of the method for non-genetic modification of microorganisms using protein ionic liquids.
Method for non-genetic transfection of microorganisms using water-free protein ionic liquids
The method comprises inoculating growth media with specified microorganisms, growing to desired density, harvesting cells, synthesizing protein ionic liquids by cationizing proteins like GFP, balancing with poly(ethylene glycol) 4-nonylphenyl 3-sulfopropyl ether potassium salt anions, lyophilizing to remove water, warming to form liquid, reconstituting, mixing with cells, suspending, freezing between −20 to −212 °C, and removing water to make a cell powder.
Reconstitution and preparation of transfected cells
Reconstituting the cell powder in 0.1 M Tris HCl buffer and mixing to obtain a uniform suspension, followed by centrifugation to obtain a cell pellet.
Washing steps for removal of excess reagents
Washing the cell pellet with 0.1 M heparin solution and pelleting, followed by washing with 0.1 M Tris HCl and pelleting to remove free protein ionic liquid.
Growth conditions for microorganisms
Inoculating sterile LB media with E. coli DH5α, C. lytica, or B. subtilis and growing at about 37 °C, or inoculating TSB media with Pichia pastoris and growing at about 30 °C.
Harvesting conditions
Harvesting cells by centrifugation at 4500-8500 rpm for 5 minutes and removing supernatant to obtain cell pellets.
Freezing and drying steps for transfection
Freezing cells suspended in protein ionic liquid solution at −20 to −212 °C in freezer or by liquid nitrogen immersion for 2-20 minutes, followed by removal of water by lyophilization or vacuum concentration under vacuum.
Post-lyophilization processing of transfected cells
Reconstituting the lyophilized cell powder in Tris HCl buffer, mixing to form suspension, centrifuging at 4500-8500 rpm for 2-10 minutes to obtain cell pellet, and removing supernatant.
The claims cover a comprehensive method for synthesizing water-free protein ionic liquids and using them for non-genetic intracellular modification of various microorganisms by growing, harvesting, treating with protein ionic liquids, freezing, lyophilizing, reconstitution, washing, and recovery of cells. These inventive features establish a universal, temporary, intracellular protein delivery technique avoiding permanent genetic modification.
Stated Advantages
Enables non-genetic, temporary modification of microorganisms without permanent genetic changes, eliminating risks of GMO release.
Provides a universal, inexpensive, general protein transfection reagent compatible with gram-negative bacteria, yeast, and spores.
Allows intracellular delivery of functional proteins of various sizes and geometries, including simultaneous multicomponent transfections.
Enhances protein stability and protection from proteases inside the cytoplasm via protein ionic liquid stabilization.
Facilitates applications such as synthetic biology, living inks, cell ruggedization, and preservation under extreme conditions.
Method exhibits minimal cell viability loss (typically under 15%), maintaining microbial function post-transfection.
Documented Applications
Temporary manipulation of cell phenotype and proteome by intracellular protein loading.
Modification and neutralization of bacterial spores.
Ruggedization of microorganisms for preservation and storage under extreme environmental conditions.
Printing of living inks with minimal loss of cell viability.
Delivery of optically, catalytically, or magnetically responsive nanomaterials inside microorganisms for biosensing.
Synthetic biology applications involving temporary control of cell function and introduction of new cellular processes.
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