Methods for making ribosomes

1. A platform for preparing a sequence defined biopolymer in vitro, the platform comprising:

(a) a ribosome-depleted cellular extract comprising an S150 bacterial extract harvested from mid-exponential growth phase to late-exponential growth phase at an OD 600 greater than 3 and up to 5, the ribosome-depleted cellular extract having a protein concentration of greater than about 5 mg/ml, the ribosome-depleted cellular extract having a polyamine at a concentration of 1.0-5.0 mM, and the ribosome-depleted cellular extract having a concentration of salts from about 50 mM to about 300 mM;

(b) ribosomal RNAs prepared by in vitro transcription; and

(c) purified ribosomal proteins depleted of ribosomal RNAs.

2. The platform according to claim 1 , wherein the polyamine is selected from the group consisting of spermine, spermidine and putrescine.

3. The platform according to claim 1 , further comprising at least one exogenous DNA template encoding ribosomal RNAs and at least one exogenous DNA template encoding a mRNA for the sequence defined biopolymer.

4. The platform according to claim 1 , wherein the ribosomal RNAs are prepared from an isolated nucleic acid comprising SEQ ID NO: 26, or variants thereof.

5. The platform according to claim 1 , wherein the ribosomal RNAs comprise transcripts produced from one or more isolated nucleic acids.

6. The platform according to claim 1 , wherein ribosomes assemble from the ribosomal RNA and the ribosomal proteins to produce biopolymers.

7. The platform according to claim 1 , wherein the platform is configured for fed-batch operation or continuous operation.

8. The platform according to claim 1 , wherein at least one substrate is replenished.

9. The platform according to claim 1 , further comprising a DNA-dependent RNA polymerase.

10. The platform according to claim 1 , further comprising at least one macromolecular crowding or volume-excluding agent.

11. The platform according to claim 1 , further comprising at least one reducing agent.

12. A method for preparing a sequence defined biopolymer in vitro, the method comprising:

(a) providing the platform according to claim 1 ;

(b) adding the purified ribosomal proteins depleted of ribosomal RNA to the ribosomal RNAs prepared by in vitro transcription in the presence of the ribosome-depleted extract to provide a translation platform mixture; and

(c) providing an RNA transcription template encoding the sequence defined biopolymer to the translational platform mixture to prepare the sequence defined biopolymer in vitro.

13. A method for preparing a sequence defined biopolymer in vitro, the method comprising:

(a) providing a translation platform mixture, wherein the translation platform mixture is prepared from a platform of claim 1 by adding the purified ribosomal proteins depleted of ribosomal RNA of part (c) to the ribosomal RNAs prepared by in vitro transcription of part (b) in the presence of the ribosome-depleted extract of part (a); and

(b) providing an RNA transcription template encoding the sequence defined biopolymer to the translation platform mixture to prepare the sequence defined biopolymer in vitro.

14. A platform for preparing a sequence defined biopolymer in vitro, the platform comprising:

(a) a ribosome-depleted cellular extract prepared from an S150 bacterial extract harvested at about 3 OD 600 , the ribosome-depleted cellular extract having a protein concentration of greater than about 5 mg/ml, the ribosome-depleted cellular extract having a polyamine at a concentration of 1.0-5.0 mM, and the ribosome-depleted cellular extract having a concentration of salts from about 50 mM to about 300 mM;

(b) ribosomal RNAs prepared by in vitro transcription; and

purified ribosomal proteins depleted of ribosomal RNAs.

15. A platform for preparing a sequence defined biopolymer in vitro, the platform comprising:

(a) a ribosome-depleted cellular extract comprising an S150 bacterial extract harvested from mid-exponential growth phase to late-exponential growth phase at an OD 600 greater than 3 and up to 5, the ribosome-depleted cellular extract having a protein concentration of greater than about 5 mg/ml, the ribosome-depleted cellular extract having a polyamine at a concentration of 1.0-5.0 mM, and the ribosome-depleted cellular extract having a concentration of salts from about 50 mM to about 300 mM;

(b) ribosomal RNAs prepared by in vitro transcription, wherein the ribosomal RNAs are transcribed from an rRNA-encoding template comprising a synthetic 3′ gene modification that enables highly efficient termination of the transcribed ribosomal RNAs; and

(c) purified ribosomal proteins depleted of ribosomal RNAs.

16. The platform of claim 15 , wherein the synthetic 3′ gene modification is a hammerhead ribozyme.

17. The platform of claim 15 , wherein the synthetic 3′ gene modification is a transcription terminator.

18. A platform for preparing a sequence defined biopolymer in vitro, the platform comprising:

(a) a ribosome-depleted cellular extract comprising an S150 bacterial extract harvested from mid-exponential growth phase to late-exponential growth phase at an OD 600 greater than 3 and up to 5, the ribosome-depleted cellular extract having a protein concentration of greater than about 5 mg/ml, the ribosome-depleted cellular extract having a polyamine at a concentration of 1.0-5.0 mM, and the ribosome-depleted cellular extract having a concentration of salts from about 50 mM to about 300 mM;

(b) ribosomal RNAs prepared by in vitro transcription, wherein the ribosomal RNAs are transcribed from an rRNA-encoding template comprising native operon structure and RNA processing sites that enhance synthesis and stoichiometric balancing of the rRNA; and

(c) purified ribosomal proteins depleted of ribosomal RNAs.

19. A method for preparing a sequence defined biopolymer in vitro, the method comprising:

(a) providing the platform according to claim 15 ;

(b) adding the purified ribosomal proteins depleted of ribosomal RNA to the ribosomal RNAs prepared by in vitro transcription in the presence of the ribosome-depleted extract to provide a translation platform mixture; and

(c) providing an RNA transcription template encoding the sequence defined biopolymer to the translational platform mixture to prepare the sequence defined biopolymer in vitro.

20. A method for preparing a sequence defined biopolymer in vitro, the method comprising:

(a) providing the platform according to claim 18 ;

(b) adding the purified ribosomal proteins depleted of ribosomal RNA to the ribosomal RNAs prepared by in vitro transcription in the presence of the ribosome-depleted extract to provide a translation platform mixture; and

(c) providing an RNA transcription template encoding the sequence defined biopolymer to the translational platform mixture to prepare the sequence defined biopolymer in vitro.