Devices and methods for producing nucleic acids and proteins

Abstract

The present disclosure generally relates to devices, compositions and methods for designing and producing nucleic acid molecules and the production of encoded proteins using these nucleic acid molecules. In some aspect, the disclosure relates to automation for the in vitro generation of coding DNA molecules, the in vitro transcription of these DNA molecules to generate protein coding RNA molecules, and the in vitro translation of these protein coding RNA molecules to produce proteins.

1 . A method of producing a protein, the method comprising:
 (a) designing a nucleic acid molecule encoding the protein,   (b) generating oligonucleotides encoding subportions of the nucleic acid molecule,   (c) assembling the oligonucleotides to produce a population of nucleic acid molecules encoding the protein,   (d) contacting the population of nucleic acid molecules encoding the protein with a first mixture suitable for the in vitro transcription and translation of members of the population of nucleic acid molecules encoding the protein to form a second mixture, and   (e) incubating the second mixture of (d) under conditions suitable for the production of (1) mRNA encoding the protein and (2) the protein,   wherein the first mixture suitable for the in vitro transcription and translation of members of the population of nucleic acid molecules comprises cellular components from cells of a first organism,   wherein some or all of the codons of the nucleic acid molecules encoding the protein are optimized for translation in a second organism, and   wherein the first organism and the second organism are of different species.   
     
     
         2 . The method of  claim 1 , wherein the population of nucleic acid molecules encoding the protein are linear. 
     
     
         3 . The method of  claim 2 , wherein the linear nucleic acid molecules comprise a promoter operable linked to the protein coding regions. 
     
     
         4 .- 5 . (canceled) 
     
     
         6 . The method of  claim 1 , wherein the second mixture of (d) contains transfer RNA molecules obtained from an organism other than the first organism. 
     
     
         7 . The method of  claim 1 , wherein the second mixture of (d) contains transfer RNA from the second organism. 
     
     
         8 .- 9 . (canceled) 
     
     
         10 . The method of  claim 1 , wherein some or all of the codons of the nucleic acid molecule encoding the protein are optimized for translation in cells of the second organism are selected to have a GC content below 55%. 
     
     
         11 . (canceled) 
     
     
         12 . The method of  claim 1 , wherein the second organism is  Saccharomyces cerevisiae.    
     
     
         13 . The method of  claim 1 , wherein at least half of the population of nucleic acid molecules encoding are the most preferred codons  Saccharomyces cerevisiae.    
     
     
         14 . (canceled) 
     
     
         15 . The method of  claim 1 , wherein expression of nucleic acid molecules containing codons for preferred codons of the first organism results in the production of less protein than expression of nucleic acid molecules containing codons for preferred codons of the second organism and wherein the difference in protein production averages greater than 10%. 
     
     
         16 . The method of  claim 1 , wherein transfer RNA molecules are exogenously added to the first mixture suitable for the in vitro transcription and translation. 
     
     
         17 . The method of  claim 16 , wherein the first mixture suitable for the in vitro transcription and translation is obtained from a mammalian cell. 
     
     
         18 . The method of  claim 17 , wherein the cell is a yeast cell. 
     
     
         19 .- 26 . (canceled) 
     
     
         27 . A reaction mixture comprising:
 (a) a cell extract obtained from mammalian cells,   (b) a nucleic acid molecule encoding a protein, wherein at least half of the codons encoding the protein are not preferred codons of the mammalian cells, and   (c) transfer RNA molecules obtained from non-mammalian cells;   wherein the average GC content of the codons encoding the protein is less than 50%.   
     
     
         28 . The reaction mixture of  claim 27 , wherein the mammalian cell is a human cell. 
     
     
         29 . The reaction mixture of  claim 28 , wherein the human cell is a HeLa cell. 
     
     
         30 . The reaction mixture of  claim 27 , wherein the population of nucleic acid molecules encoding the protein are linear. 
     
     
         31 . The reaction mixture of  claim 30 , wherein the linear nucleic acid molecules comprise a promoter operable linked to the protein coding regions. 
     
     
         32 . The reaction mixture of  claim 30 , wherein the mammalian cell is a mouse cell. 
     
     
         33 . (canceled) 
     
     
         34 . A mechanical device comprising:
 (a) a control device capable of performing the following functions:
 (1) accepting the input of nucleotide or amino acid sequence data, 
 (2) designing nucleic acid molecules corresponding to the input nucleotide sequence or encoding the amino acid sequence, 
 (3) directing reagent flows and incubation times for the synthesis and assembly of nucleic acid molecules designed in (2), and 
 (4) directing reagent flows and incubation times for the in vitro transcription and translation of nucleic acid molecules synthesized by the device using the reagent flows and incubation times for directed in (3), 
   (b) one or more reagent storage reservoirs,   (c) one or more reaction sites for the synthesis and assembly of nucleic acid molecules and the in vitro transcription and translation of nucleic acid molecules to produce proteins,   (d) one or more ports for the removal of a nucleic acid molecules and/or proteins produced by the device, and   (e) optionally, one or more means for detecting and/or purifying and/or quantifying the proteins produced by the device.   
     
     
         35 .- 36 . (canceled) 
     
     
         37 . The device of  claim 34 , wherein one or more reagent reservoirs contain one or more reagent selected from the group consisting of:
 (a) a washing solution,   (b) a mis-match repair endonuclease,   (c) a cellular extract suitable for in vitro transcription and/or in vitro translation, and   (d) one or more nucleoside phosphoramidite.   
     
     
         38 .- 64 . (canceled)