Genetically modified biological cells
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 and 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 couple.”
1. 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 B12, which is the enzyme with EC 1.3.1.54 or EC 2.1.1.133, wherein the expression of the positive selectable marker is necessary for survival of the genetically modified bacterial cell in media lacking said vitamin, and wherein promoter of the expression cassette drives expression of positive selectable marker and expression of the protein of interest.
2. The recombinant genetically modified bacterial cell of claim 1 , wherein the cell is a cyanobacterium.
3. The recombinant genetically modified bacterial cell of claim 2 , 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, Cylindrospermum 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.
4. The recombinant genetically modified bacterial cell of claim 1 , wherein the protein of interest is a metal-binding protein.
5. The recombinant genetically modified bacterial cell of claim 4 , wherein the metal-binding protein is selected from the group consisting of a metallothionein and a transcription factor.
6. The recombinant genetically modified bacterial cell of claim 1 , wherein the protein of interest is a protease, an oxidase, a phytase, a chitinase, an invertase, a lipase, a cellulase, a xylenase, a kinase, or a phosphatase.
7. The recombinant genetically modified bacterial cell of claim 1 , wherein the enzyme is pyruvate dehydrogenase, dihydrolipoyl dehydrogenase, dihydrolipoyllysine-residue acetyltransferase, acteyl-CoA carboxylase, malonyl-CoA:ACP transacylase or fatty acid synthase.
8. The recombinant genetically modified bacterial cell of claim 1 , wherein the recombinant expression cassette further comprises a nucleic acid molecule encoding a purification tag operatively coupled to the first nucleic acid molecule.
9. The recombinant genetically modified bacterial cell of claim 1 , wherein the recombinant expression cassette further comprises a nucleic acid molecule encoding a linker between the protein of interest and the selectable marker protein, wherein said linker acts as a hinge and wherein said protein of interest and the selectable marker enzyme retain biological activity.
10. The recombinant genetically modified bacterial cell of claim 1 , wherein the protein of interest is a lipase.
11. A kit comprising the bacterial cell of claim 1 and instructional materials.