Methods for making and using genomically recoded cells
Methods making and using genomically recoded cells or organisms are provided including genomically recoded cells or organisms that lack the ability to translate a foreign nucleic acid sequence into a polypeptide that may be toxic to the genomically recoded cell or organism.
1 . A method for creating a population of prokaryotic cells comprising a DNA sequence of canonical codons encoding a polypeptide toxic to the prokaryotic cells comprising
introducing into an initial prokaryotic cell the DNA sequence of canonical codons encoding the polypeptide toxic to the initial prokaryotic cell,
wherein the initial prokaryotic cell is genomically recoded and lacks expression of one or more tRNAs cognate to one or more canonical codons thereby rendering the one or more canonical codons unassigned in the cell,
wherein lack of the initial prokaryotic cell to produce the one or more tRNAs cognate to the one or more canonical codons prevents translation and expression of the DNA sequence into the polypeptide toxic to the initial prokaryotic cell,
wherein the initial prokaryotic cell is grown to produce a population of prokaryotic cells in which the DNA sequence is stably maintained and propagated in the prokaryotic cells while translation and expression of the DNA sequence is prevented by the absence of a cognate tRNA.
2 . The method of claim 1 wherein the cell is a bacterial cell.
3 . The method of claim 1 wherein the polypeptide is a protein.
4 . The method of claim 1 , wherein toxicity is determined by a reduction in prokaryotic cell growth rate by at least 1% compared to growth rate of a corresponding wild type prokaryotic cell.
5 . The method of claim 1 wherein the polypeptide is a protein that is toxic to the prokaryotic cell.
6 . The method of claim 1 wherein the polypeptide is a protein that is toxic to the prokaryotic cell and wherein lack of the prokaryotic cell to produce the one or more tRNAs cognate to the one or more canonical codons prevents expression of the DNA sequence into the polypeptide, thereby reducing toxicity to the prokaryotic cell of the polypeptide compared to a non-recoded prokaryotic cell of the same strain.
7 . The method of claim 1 wherein one or more tRNAs cognate to the one or more unassigned codons are provided within the prokaryotic cells of the population, and wherein the prokaryotic cells of the population express the DNA sequence into the polypeptide.
8 . The method of claim 1 further comprising
providing the prokaryotic cell with an expression plasmid or vector that is expressed to provide the one or more tRNAs cognate to the one or more canonical codons within the DNA sequence, wherein the prokaryotic cells within the population of prokaryotic cells transcribe and translate the DNA sequence to produce the polypeptide, or
wherein the prokaryotic cells within the population of prokaryotic cells are genetically modified to encode one or more tRNAs cognate to one or more canonical codons within the DNA sequence under the influence of an inducible promoter and inducing the inducible promoter to begin transcription and translation of the DNA sequence to produce the polypeptide.
9 . The method of claim 1 , wherein toxicity is determined by a reduction in prokaryotic cell growth rate by at least 10% compared to growth rate of a corresponding wild type prokaryotic cell.
10 . The method of claim 1 , wherein toxicity is determined by a reduction in prokaryotic cell growth rate by at least 50% compared to growth rate of a corresponding wild type prokaryotic cell.
11 . The method of claim 1 , wherein toxicity is determined by a reduction in prokaryotic cell growth rate by at least 90% compared to growth rate of a corresponding wild type prokaryotic cell.
12 . The method of claim 1 wherein the initial genomically recoded prokaryotic cell also lacks expression of one or more release factors cognate to one or more canonical codons.
13 . A population of genomically recoded prokaryotic cells wherein the prokaryotic cells of the population lack expression of one or more tRNAs cognate to one or more canonical codons, wherein the prokaryotic cells within the population of prokaryotic cells comprise a DNA sequence of canonical codons encoding a polypeptide toxic to the prokaryotic cells, and wherein DNA sequence comprises the canonical codons whose recognition by the one or more cognate tRNAs is absent due to the lack of expression of the one or more cognate tRNAs,
wherein the population of genomically recoded prokaryotic cells is grown from a genomically recoded prokaryotic cell including the DNA sequence of canonical codons encoding the polypeptide toxic to the prokaryotic cell, and wherein the DNA sequence is stably maintained and propagated in the prokaryotic cells while translation and expression of the DNA sequence is prevented by the absence of a cognate tRNA.
14 . The population of genomically recoded prokaryotic cells of claim 13 wherein the prokaryotic cells of the population also lack expression of one or more release factors cognate to one or more canonical codons.
15 . A population of altered prokaryotic cells lacking expression of one or more tRNAs cognate to one or more canonical codons within a DNA sequence encoding a polypeptide toxic to the prokaryotic cells comprising
the DNA sequence,
an expression plasmid or vector encoding the one or more tRNAs cognate to the one or more canonical codons within the DNA sequence,
wherein the DNA sequence comprises the canonical codons whose recognition by the one or more cognate tRNAs is absent due to the lack of expression of the one or more cognate tRNAs, and
wherein the prokaryotic cell is genomically recoded, and
wherein the DNA sequence is stably maintained and propagated in the prokaryotic cells while translation and expression of the DNA sequence is prevented by the absence of a cognate tRNA.
16 . The population of altered prokaryotic cells of claim 15 further lacking expression of one or more release factors cognate to one or more canonical codons.