Genetically Modified Biological Cells

Abstract

The present invention is based, in part, on our discovery of a way to configure expression cassettes so that the expression of a selectable marker protein, which is critical for the growth or survival of a cell, also results in the expression of a protein of interest in a biological cell. Accordingly, in one aspect, the invention features a genetically modified cell (e.g., a bacterial cell) that includes a chromosomally integrated or cytoplasmic expression cassette that includes a first nucleic acid sequence encoding a protein of interest an a second nucleic acid sequence encoding a selectable marker protein. The regulatory sequence (e.g., the sequence encoding a functional promoter) that drives expression of the required selectable marker protein also drives expression of the protein of interest. For that reason, we may refer to their expression as being “linked” or “functionally coupled.”

1 - 63 . (canceled) 
     
     
         64 . A recombinant genetically modified bacterial cell comprising:
 a recombinant chromosomally integrated expression cassette, the recombinant chromosomally integrated expression cassette comprising a first nucleic acid molecule encoding a protein of interest and a second nucleic acid molecule encoding a positive selectable marker, said positive selectable marker being an enzyme necessary for the production of an essential vitamin,
 wherein the expression of the positive selectable marker is necessary for survival of the genetically modified bacterial cell in media lacking the essential vitamin; 
 and wherein promoter of the expression cassette drives expression of the positive selectable marker and expression of the protein of interest. 
   
     
     
         65 . The recombinant genetically modified bacterial cell of  claim 64 , wherein the recombinant expression cassette further comprises a nucleic acid molecule encoding a purification tag functionally coupled to the first nucleic acid molecule. 
     
     
         66 . The recombinant genetically modified bacterial cell of  claim 64 , wherein the recombinant expression cassette further comprises a nucleic acid molecule encoding a linker between the protein of interest and the selectable marker protein. 
     
     
         67 . The recombinant genetically modified bacterial cell of  claim 64 , wherein the essential vitamin is vitamin B12. 
     
     
         68 . The recombinant genetically modified bacterial cell of  claim 67 , wherein the positive selectable marker is an enzyme necessary for the production of vitamin B12. 
     
     
         69 . The recombinant genetically modified bacterial cell of  claim 68 , wherein the enzyme necessary for the production of vitamin B12 is an enzyme with an enzyme classification of EC 2.1.1.130, EC 1.14.13.83, EC 2.1.1.131, EC 2.1.1.152, EC 2.1.1.132, EC 5.4.99.61, EC 6.3.5.9, EC 6.6.1.2, EC 2.5.1.17, EC 6.3.5.10, EC 2.7.7.62, EC 2.7.8.26, EC 4.99.1.3, EC 2.1.1.151, EC 2.1.1.271, EC 2.1.1.195, EC 2.1.1.196, EC 6.3.5.11, EC 1.3.1.54, or EC 2.1.1.133. 
     
     
         70 . The recombinant genetically modified bacterial cell of  claim 64 , wherein the essential vitamin is ascorbic acid (vitamin C), biotin (vitamin B7), choline, folic acid (vitamin B9), inositol, nicotinic acid/niacin (vitamin B3), para-aminobenzoic acid (PABA), pantothenic acid (vitamin B5), pyridoxine (vitamin B6), riboflavin (vitamin B2), thiamine (vitamin B1), thymidine retinoid (vitamin A), tocopherol (vitamin E), naphthoquinon (vitamin K), ergocalciferol (vitamin D), or the pantothenic acid derivative coenzyme A. 
     
     
         71 . The recombinant genetically modified bacterial cell of  claim 64 , wherein the protein of interest is selected from the group consisting of a bacterial protein, a plant protein, and a mammalian protein, an insect protein, a fish protein, and a fungal protein. 
     
     
         72 . The recombinant genetically modified bacterial cell of  claim 64 , wherein the protein of interest is a metal-binding protein. 
     
     
         73 . The recombinant genetically modified bacterial cell of  claim 72 , wherein the metal-binding protein is selected from the group consisting of a metallothionein and a transcription factor. 
     
     
         74 . The recombinant genetically modified bacterial cell of  claim 64 , wherein the protein of interest is an enzyme. 
     
     
         75 . The recombinant genetically modified bacterial cell of  claim 64 , wherein the protein of interest is a protease, an oxidase, a phytase, a chitinase, an invertase, a lipase, a cellulase, a xylenase, a kinase, a phosphatase, or an enzyme within a biosynthetic pathway that results in the production of an oil. 
     
     
         76 . The recombinant genetically modified bacterial cell of  claim 64 , wherein the protein of interest is an enzyme active in the synthesis of fatty acids 10-16 carbons in length, an enzyme that accelerates glycerol metabolism, or an enzyme that converts lactic acid to pyruvate. 
     
     
         77 . The recombinant genetically modified bacterial cell of  claim 64 , wherein the protein of interest is an enzyme active in the synthesis of fatty acids 10-16 carbons in length is pyruvate dehydrogenase, dihydrolipoyl dehydrogenase, dihydrolipoyllysine-residue acetyltransferase, acteyl-CoA carboxylase, malonyl-CoA:ACP transacylase or fatty acid synthase. 
     
     
         78 . The recombinant genetically modified bacterial cell of  claim 64 , wherein the protein of interest is selected from the group consisting of a metal-binding protein, an enzyme, an immunogen, a toxin, an anti-toxin, a prolamine, zein, a hormone, a transcription factor, a growth factor, an anticoagulant, a lysosomal protein, an enzyme substrate, a receptor or a subunit thereof, a heavy chain of an immunoglobulin, a light chain of an immunoglobulin, and a single chain antibody. 
     
     
         79 . The recombinant genetically modified bacterial cell of  claim 64 , wherein the protein of interest is a lipase. 
     
     
         80 . The recombinant genetically modified bacterial cell of  claim 64 , wherein the cell is a cyanobacterium cell. 
     
     
         81 . The recombinant genetically modified bacterial cell of  claim 80 , wherein the cyanobacterium is  Anabaena  species (sp),  Anabaenopsis  sp,  Aphanizomenon  sp,  Arthrospira  sp,  Calothrix  sp,  Chamaesiphon  sp,  Chlorogloeopsis  sp,  Chroococcidiopsis  sp,  Chroococcus  sp,  Cyanothece  sp, Cyhndrospermum sp,  Dactylococcopsis  sp,  Dermocarpella  sp,  Fischerella  sp,  Geitlerinema  sp,  Gloeobacter  sp,  Gloeocapsa  sp,  Gloeothece  sp,  Leptolyngbya  sp,  Lyngbya  sp,  Microchaete  sp,  Microcoleus  sp,  Microcystis  sp,  Myxosarcina  sp,  Nodularia  sp,  Nostoc  sp,  Oscillatoria  sp,  Pleurocapsa  sp,  Pseudanabaena  sp,  Scytonema  sp,  Spirulina  sp,  Stanieria  sp,  Symploca  sp,  Synechococcus  sp,  Synechocystis  sp,  Tolypothrix  sp, or  Xenococcus  sp. 
     
     
         82 . A kit comprising the recombinant genetically modified bacterial cell of  claim 64 .