WO2020033416A1 - Combined transcription and translation platform derived from plant chloroplasts - Google Patents

Abstract

Disclosed are compositions, methods, and kits for performing cell-free protein synthesis (CFPS). The disclosed compositions, methods, and kits include or utilize components prepared from plant chloroplasts or extracts thereof. The compositions, methods, and kits may be used for in vitro protein synthesis and prototyping of genetic expression in plants and are suitable for automation.

Description

COMBINED TRANSCRIPTION AND TRANSLATION PLATFORM

DERIVED FROM PLANT CHLOROPLASTS

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR

DEVELOPMENT

[0001] This invention was made with government support under HR0011-15-C-

0084 awarded by the Defense Advanced Research Projects Agency (DARPA). The government has certain rights in the invention.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

[0002] The present application claims the benefit of priority under 35 U.S.C. §

119(e) to U.S. Provisional Application No. 62/772,341, filed on November 28, 2019, and to U.S. Provisional Application No. 62/714,916, filed on August 6, 2019, the contents of which are incorporated herein by reference in their entireties.

BACKGROUND

[0003] The present invention generally relates to compositions, methods, and kits for performing cell-free protein synthesis (CFPS). More specifically, the present invention relates to compositions, methods, and kits for performing cell-free protein synthesis (CFPS) that include or utilize components prepared from plant chloroplasts or extracts thereof. The compositions, methods, and kits may be used for in vitro protein synthesis and prototyping of genetic expression in plants.

[0004] Cell-free systems have recently enjoyed a technical renaissance that has transformed them into robust platforms for the synthesis of a wide variety of useful and interesting products [1-4] Such platforms combine crude cell lysates or purified components with substrates in a test tube, enabling the activation and use of cellular processes in vitro. Cell-free protein synthesis (CFPS) systems in particular have made significant advances in reaction volume, duration, and productivity, now reaching g/L quantities of protein[3, 5-12] These systems provide several unique advantages for understanding, harnessing, and expanding the capabilities of natural biological systems. Reactions are open, and are therefore easily accessible for sample extraction and substrate feeding. Dilute reaction environments facilitate the folding of complex eukaryotic protein products which may otherwise express poorly in bacterial systems [4] Importantly, the removal of genomic material from the chassis organism directs reaction substrates and machinery towards the desired synthesis reaction at high rates. Exploiting these features, CFPS platforms enjoy increasingly widespread use as a complement to in vivo expression for applications including biomolecular breadboarding[l3-l6], expression of toxic products [17-20], production of complex protein products that are poorly soluble in vivo[ 7, 21-23], manufacture of glycoproteins[24, 25], detection of disease[26-28], and on demand biomanufacturing[2l, 29-32]

[0005] Despite the emergence of cell-free systems as a prominent research tool for fundamental and applied biology, the vast majority of previous efforts have focused on a select few model systems such as Escherichia coli, Saccharomyces cerevisiae, and Chinese Hamster Ovary cells, among others[2, 5, 11, 12, 33, 34] However, we and others hypothesize that developing cell-free systems composed of extracts derived from relevant chassis organisms that better mimic the natural physicochemical environment might enhance predictive power for synthetic biology applications. This idea motivates the development of new cell-free systems. In this context, several new CFPS systems have been developed, including some from Streptomyces species and Bacillus [35-39] For example, an elegant study by Freemont and colleagues showed characterized new DNA parts from the non-model bacterium Bacillus megaterium by combining automated CFPS and Bayesian models[39]

[0006] A particularly exciting chassis organism for developing a new cell-free system is plant chloroplasts. Unfortunately, the current state of the art in transformation of plant chloroplasts is a laborious, low-throughput method that takes approximately one year to produce a stably transgenic plant. As a result, developments in plant biotechnology and our understanding of the basic biology of plants have lagged far behind what we have been able to achieve with mammalian or prokaryotic biology, in spite of the fact that plants are considered to be the source of a rich potential for pharmaceutically and technologically relevant natural products. Developing a cell-free system that could be used to prototype genetic design and establish part libraries for plants could be transformative. Indeed, new genetic parts (e.g., promoters, ribosome binding sites, terminators) could facilitate forward engineering.

[0007] Here, we disclose such a cell-free system using components prepared from plant chloroplasts and extracts from Nicotiana tabacum as a model. Our disclosed cell- free system can be applied to prepare similar cell-systems using components prepared from plant chloroplasts and extracts from other plants in order to prototype genetic parts and establish part libraries.

SUMMARY

[0008] Disclosed are compositions, methods, and kits for performing cell-free protein synthesis (CFPS). The disclosed compositions, methods, and kits include or utilize components prepared from plant chloroplasts and extracts thereof. The compositions, methods, and kits may be used for in vitro protein synthesis and prototyping of genetic expression in plants. The methods disclosed herein are suitable for automation.

BRIEF DESCRIPTION OF THE FIGUREES

[0009] Figure 1 provides an overview of the isolation of intact chloroplasts from plant material and preparation of an extract from the isolated chloroplasts for use in cell- free transcription and/or translation.

[0010] Figure 2 illustrates the relative protein concentration of ribulose-1,5- bisphosphate carboxylase/oxygenase (Rubisco), an enzyme present in chloroplasts, versus the concentration of membrane proteins in a extract as an indicator of intact chloroplasts versus broken chloroplasts. [0011] Figure 3 illustrates the production of mRNA in a cell-free system comprising a chloroplast extract as a function of the concentration of a DNA template for mRNA present in the system.

[0012] Figure 4 illustrates the results of experiments in which a template DNA encoding a fluorescent target protein was transcribed and translated in a cell-free systems comprising a chloroplast extract.

[0013] Figure 5 illustrates that divalent cations such as Mg²⁺ are required for efficient protein production in a cell-free system comprising a chloroplast extract in which a template DNA encoding a fluorescent target protein was transcribed and translated as in Figure 4.

[0014] Figure 6 illustrates that extracts prepared from chloroplasts isolated from plants that were grown at six (6) weeks versus four (4) weeks exhibit better performance regarding protein production in a cell-free free system.

[0015] Figure 7 illustrates a method to increase lysate activity related to total protein content by e.g. dark incubation of plants before extract preparation a) Light conditions during normal growth and dark incubation of plants before chloroplast extract purification. After 6-24h of dark incubation starch granules are reduced in the chloroplasts (light microscopic images) which results in b) higher total protein content (measured with Bradford assay) and lysate productivity (measured as expressed luciferase) in the chloroplast cell-free system. SD from technical replicates of purified lysates.

[0016] Figure 8 illustrates the results of semi-continuous and batch reaction of extracts from light/dark incubated plants (2 replicates: performed with two different extracts).

[0017] Figure 9 illustrates that the addition of a polyol such as glycerol to isolated chloroplasts prior to lysis by freezing improves productivity of the lysates. [0018] Figure 10 illustrates the results of combined optimization on protein production in a cell-free system comprising a chloroplast extract, including addition of a polyol prior to lysis of chloroplasts to prepare the extract, timing of growth in plants from which the chloroplast extract is prepared, and codon optimization for expression in the plants from which the chloroplast extract is prepared.

[0019] Figure 11 illustrates that protein expression is influenced by modification in the 5'-UTR and 3'-UTR of the expression template.

[0020] Figure 12 illustrates the effect of magnesium acetate versus magnesium glutamate on protein production in a cell-free system comprising a chloroplast extract.

[0021] Figure 13 illustrates the effect of adding potassium on protein production in a cell-free system comprising a chloroplast extract.

[0022] Figure 14 illustrates the effect of pH in the presence of a HEPES buffered system versus a Tris buffered system on protein production in a cell-free system comprising a chloroplast extract.

[0023] Figure 15 illustrates the effect of adding ammonium on protein production in a cell-free system comprising a chloroplast extract.

[0024] Figure 16 illustrates the effect of amino acid concentration on protein production in a cell-free system comprising a chloroplast extract.

[0025] Figure 17 illustrates the effect of HEPES concentration at a pH of 7.3 on protein production in a cell-free system comprising a chloroplast extract.

[0026] Figure 18 illustrates the effect of different energy regeneration systems on protein production in a cell-free system comprising a chloroplast extract, including a phosphoenolpyruvate (PEP) system, a PEP/ pyruvate kinase (PyK) system, and a creatine phosphate (CP)/ creatine kinase (CK) system. [0027] Figure 19 illustrates the effect of different concentrations of a creatine phosphate (CP)/ creatine kinase (CK) energy regeneration system on protein production in a cell-free system comprising a chloroplast extract.

[0028] Figure 20 illustrates the effect of different concentration of T7 DNA- dependent RNA-polymerase (T7 RNAP) on protein production in a cell-free system comprising a chloroplast extract.

[0029] Figure 21 illustrates the effect of final DNA template concentration at two different concentrations of T7 RNAP on protein production in a cell-free system comprising a chloroplast extract.

[0030] Figure 22 illustrates the effect of different macromolecular crowding agents on protein production in a cell-free system comprising a chloroplast extract, including Ficoll 400, PEG 600, PEG 3350, and PEG 8000.

[0031] Figure 23 illustrates the effect of different macromolecular crowding agents

(Ficoll 400, PEG 3350, and PEG 8000) at different concentrations (0.50%, 1%, 2%, and 4%) on protein production in a cell-free system comprising a chloroplast extract.

[0032] Figure 24 illustrates the effect of chloroplast extract concentration on protein production in a cell-free system comprising the chloroplast extract.

[0033] Figure 25 illustrates protein production versus time in two different cell- free systems comprising a chloroplast extract.

[0034] Figure 26 shows the effect of RNAse inhibitor on chloroplast cell-free reaction. 0, 0.5 and 1 U/pL RNAse inhibitor was added.

[0035] Figure 27 illustrates that linear templates or plasmid DNA can be used in chloroplast cell free reactions to produce protein, with linear templates offering a slight boost to yields and a faster turnaround time than plasmids. [0036] Figure 28 illustrates different mRNA expression dynamics in chloroplast and E. coli cell-free systems. A construct (pY7lmRFPlSpA) expressing a fluorescent protein (mRFP) and a fluorescent RNA aptamer (spinach) was assayed in both chloroplast and E. coli cell-free systems.

[0037] Figure 29 provides a schematic illustration of plastid prototyping using a chloroplast cell-free system and subsequent chloroplast transformation to prepare modified plants.

[0038] Figure 30 illustrates a thermodynamic model that provides the basis for designing ribosome binding sites (RBS) that have different strengths.

[0039] Figure 31 illustrates the results of experiments in which protein expression was measured versus ribosome binding strength for a library of expression vectors having different ribosome binding sites (RBS). The proteins expressed in the system included the first 99 nucleotides of biosynthetic enzyme A (Kas) or biosynthetic enzyme B (AroG) fused to luciferase where luciferase activity was measures as an output for protein production.

[0040] Figure 32 illustrates the results of experiments in which protein expression was measured versus ribosome binding strength for a library of expression vectors having different ribosome binding sites (RBS). The proteins expressed in the system were one of biosynthetic enzyme A (Kas) or biosynthetic enzyme B (AroG) where protein production was measure via incorporation of radioactive Leucine.

[0041] Figure 33 illustrates yields of protein (GFP) in a chloroplast extract using an RBS library and a 16S rRNA sequence from the chloroplast, illustrating how a chloroplast cell-free system can be utilized to rank the efficiency of genetic parts such as RBS.

[0042] Figure 34 illustrates schematically how the effect of codon-optimization on protein production can be assessed using a chloroplast cell-free system and how an algorithm can be devised from results in a chloroplast cell-free system to provide an algorithm for codon optimization prior to chloroplast transformation of an expression vector.

[0043] Figure 35 illustrates RT-qPCR analysis of a multi-genic cluster in a chloroplast cell-free system. A cluster (Klebsiella‘ refactored’ v2.) composed of 16 genes (20kb) was used as template for chloroplast cell-free reactions. Reactions were incubated 25°C during lh and 100 pL of the reaction mix was harvested for total RNA extraction. Total RNA was assayed with RT-qPCR. All 16 genes were detected in both cell-free systems, demonstrating that the chloroplast cell-free is able to express a multi-gene cluster.

[0044] Figure 36 provides a comparison of multi-gene expression in a cell-free system per Figure 37 versus multi-gene expression in vivo.

[0045] Figure 37 illustrates RT-qPCR analysis of a multi-genic cluster in chloroplast and E. coli cell-free a) A cluster (Klebsiella‘refactored’ v2.) composed of 16 genes (20kb) was used as template for chloroplast and E. coli cell-free reactions. Reactions were incubated 25°C or 37°C during lh and 100 pL of the reaction mix was harvested for total RNA extraction b) Total RNA was assayed with RT-qPCR. All 16 genes were detected in both cell-free systems, demonstrating that the chloroplast cell-free is able to express a multi-gene cluster.

[0046] Figure 38 illustrates that there is a better correlation between predicted

RBS strength and actual yields in a cell-free system that comprises a chloroplast extract versus a cell-free system that comprise an E. coli extract.

[0047] Figure 39 Chloroplast in vivo gene expression correlates with chloroplast cell-free data a) Engineered nitrogenase (nif) clusters (Klebsiella vl.0, v2.0, and v3.2) were integrated into the tobacco plastome under T7 promoters. Upon theophylline (Theo) induction, plastome integrated T7 RNAP transcribes nif clusters b) Plasmids with nif clusters assayed in the chloroplast cell-free system. GFP under T7 promoter was expressed from the plasmid backbone for normalization. Purified T7 RNAP was added to the reactions c) RNA-seq data of transplastomic plants (left hand side) shows cluster expression with and without Theo induction (full and empty circles, respectively). Correlation between wild type Klebsiella oxytoca and transplastomic lines (right hand side) d) RT-qPCR data normalized to GFP shows cluster expression in chloroplast cell- free (left hand side). Correlation between in vivo and chloroplast cell-free data (right hand side).

DETAILED DESCRIPTION

[0048] Definitions and Terminology

[0049] The disclosed subject matter may be further described using definitions and terminology as follows. The definitions and terminology used herein are for the purpose of describing particular embodiments only, and are not intended to be limiting.

[0050] As used in this specification and the claims, the singular forms“a,”“an,” and“the” include plural forms unless the context clearly dictates otherwise. For example, the term“a component” should be interpreted to mean“one or more components” unless the context clearly dictates otherwise. As used herein, the term“plurality” means“two or more.”

[0051] As used herein, “about”, “approximately,” “substantially,” and

“significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and“approximately” will mean up to plus or minus 10% of the particular term and “substantially” and “significantly” will mean more than plus or minus 10% of the particular term.

[0052] As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being“open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms“consist” and“consisting of’ should be interpreted as being“closed” transitional terms that do not permit the inclusion of additional components other than the components recited in the claims. The term“consisting essentially of’ should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.

[0053] The phrase“such as” should be interpreted as“for example, including.”

Moreover the use of any and all exemplary language, including but not limited to“such as”, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed.

[0054] Furthermore, in those instances where a convention analogous to“at least one of A, B and C, etc.” is used, in general such a construction is intended in the sense of one having ordinary skill in the art would understand the convention (e.g.,“a system having at least one of A, B and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together.). It will be further understood by those within the art that virtually any disjunctive word and/or phrase presenting two or more alternative terms, whether in the description or figures, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase“A or B” will be understood to include the possibilities of“A” or‘B or“A and B.”

[0055] All language such as“up to,”“at least,”“greater than,”“less than,” and the like, include the number recited and refer to ranges which can subsequently be broken down into subranges as discussed above.

[0056] A range includes each individual member. Thus, for example, a group having 1-3 members refers to groups having 1, 2, or 3 members. Similarly, a group having 6 members refers to groups having 1, 2, 3, 4, or 6 members, and so forth.

[0057] The modal verb“may” refers to the preferred use or selection of one or more options or choices among the several described embodiments or features contained within the same. Where no options or choices are disclosed regarding a particular embodiment or feature contained in the same, the modal verb “may” refers to an affirmative act regarding how to make or use and aspect of a described embodiment or feature contained in the same, or a definitive decision to use a specific skill regarding a described embodiment or feature contained in the same. In this latter context, the modal verb“may” has the same meaning and connotation as the auxiliary verb“can.”

[0058] Polynucleotides and Synthesis Methods

[0059] The terms“nucleic acid” and“oligonucleotide,” as used herein, refer to polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), and to any other type of polynucleotide that is an N glycoside of a purine or pyrimidine base. There is no intended distinction in length between the terms“nucleic acid”, “oligonucleotide” and “polynucleotide”, and these terms will be used interchangeably. These terms refer only to the primary structure of the molecule. Thus, these terms include double- and single-stranded DNA, as well as double- and single- stranded RNA. For use in the present methods, an oligonucleotide also can comprise nucleotide analogs in which the base, sugar, or phosphate backbone is modified as well as non-purine or non-pyrimidine nucleotide analogs.

[0060] As used herein, a“polymerase” refers to an enzyme that catalyzes the polymerization of nucleotides. “DNA polymerase” catalyzes the polymerization of deoxyribonucleotides. Known DNA polymerases include, for example, Pyrococcus furiosus (Pfu) DNA polymerase, E. coli DNA polymerase I, T7 DNA polymerase and Thermus aquaticus (Taq) DNA polymerase, among others.“RNA polymerase” catalyzes the polymerization of ribonucleotides. The foregoing examples of DNA polymerases are also known as DNA-dependent DNA polymerases. RNA-dependent DNA polymerases also fall within the scope of DNA polymerases. Reverse transcriptase, which includes viral polymerases encoded by retroviruses, is an example of an RNA-dependent DNA polymerase. Known examples of RNA polymerase (“RNAP”) include, for example, RNA polymerases of bacteriophages ( e.g . T3 RNA polymerase, T7 RNA polymerase, SP6 RNA polymerase), and E. coli RNA polymerase, among others. The foregoing examples of RNA polymerases are also known as DNA-dependent RNA polymerase. The polymerase activity of any of the above enzymes can be determined by means well known in the art.

[0061] The term“promoter” refers to a cis-acting DNA sequence that directs RNA polymerase and other trans-acting transcription factors to initiate RNA transcription from the DNA template that includes the cis-acting DNA sequence.

[0062] As used herein, the term “sequence defined biopolymer” refers to a biopolymer having a specific primary sequence. A sequence defined biopolymer can be equivalent to a genetically-encoded defined biopolymer in cases where a gene encodes the biopolymer having a specific primary sequence.

[0063] As used herein,“expression template” refers to a nucleic acid that serves as substrate for transcribing at least one RNA that can be translated into a sequence defined biopolymer (e.g., a polypeptide or protein). Expression templates include nucleic acids composed of DNA or RNA. Suitable sources of DNA for use a nucleic acid for an expression template include genomic DNA, cDNA and RNA that can be converted into cDNA. Genomic DNA, cDNA and RNA can be from any biological source, such as a tissue sample, a biopsy, a swab, sputum, a blood sample, a fecal sample, a urine sample, a scraping, among others. The genomic DNA, cDNA and RNA can be from host cell or virus origins and from any species, including extant and extinct organisms. As used herein,“expression template” and“transcription template” have the same meaning and are used interchangeably.

[0064] In certain exemplary embodiments, vectors such as, for example, expression vectors, containing a nucleic acid encoding one or more mRNAs or reporter polypeptides and/or proteins described herein are provided. As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a“plasmid,” which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Such vectors are referred to herein as“expression vectors.” In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” can be used interchangeably. However, the disclosed methods and compositions are intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0065] In certain exemplary embodiments, the recombinant expression vectors comprise a nucleic acid sequence (e.g., a nucleic acid sequence encoding one or more mRNAs or reporter polypeptides and/or proteins described herein) in a form suitable for expression of the nucleic acid sequence in one or more of the methods described herein, which means that the recombinant expression vectors include one or more regulatory sequences which is operatively linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector,“operably linked” is intended to mean that the nucleotide sequence encoding one or more mRNAs or reporter polypeptides and/or proteins described herein is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro ribosomal assembly, transcription and/or translation system). The term“regulatory sequence” is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990).

[0066] Oligonucleotides and polynucleotides may optionally include one or more non-standard nucleotide(s), nucleotide analog(s) and/or modified nucleotides. Examples of modified nucleotides include, but are not limited to diaminopurine, S²T, 5-fluorouracil, 5- bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5- (carboxyhy droxy lmethy l)uracil, 5 -carboxy methy laminomethy l-2-thiouridine, 5 - carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6- isopentenyladenine, 1 -methy lguanine, 1 -methy linosine, 2,2-dimethylguanine, 2- methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7- methylguanine, 5 -methy laminomethy luracil, 5-methoxyaminomethyl-2-thiouracil, beta-D- mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-D46- isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2- thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5- oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino- 3-N-2-carboxypropyl) uracil, (acp3)w, 2,6-diaminopurine and the like. Nucleic acid molecules may also be modified at the base moiety (e.g., at one or more atoms that typically are available to form a hydrogen bond with a complementary nucleotide and/or at one or more atoms that are not typically capable of forming a hydrogen bond with a complementary nucleotide), sugar moiety or phosphate backbone.

[0067] Peptides Polypeptides. Proteins and Synthesis Methods

[0068] As used herein, the terms“peptide,”“polypeptide,” and“protein,” refer to molecules comprising a chain a polymer of amino acid residues joined by amide linkages. The term“amino acid residue,” includes but is not limited to amino acid residues contained in the group consisting of alanine (Ala or A), cysteine (Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E), phenylalanine (Phe or F), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), lysine (Lys or K), leucine (Leu or L), methionine (Met or M), asparagine (Asn or N), proline (Pro or P), glutamine (Gln or Q), arginine (Arg or R), serine (Ser or S), threonine (Thr or T), valine (Val or V), tryptophan (Trp or W), and tyrosine (Tyr or Y) residues. The term“amino acid residue” also may include nonstandard, noncanonical, or unnatural amino acids, which optionally may include amino acids other than any of the following amino acids: alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, and tyrosine residues. The term“amino acid residue” may include alpha-, beta-, gamma-, and delta- amino acids.

[0069] In some embodiments, the term “amino acid residue” may include nonstandard, noncanonical, or unnatural amino acid residues contained in the group consisting of homocysteine, 2-Aminoadipic acid, N-Ethylasparagine, 3-Aminoadipic acid, Hydroxylysine, b-alanine, b-Amino-propionic acid, allo-Hydroxylysine acid, 2- Aminobutyric acid, 3-Hydroxyproline, 4-Aminobutyric acid, 4-Hydroxyproline, piperidinic acid, 6-Aminocaproic acid, Isodesmosine, 2-Aminoheptanoic acid, allo- Isoleucine, 2-Aminoisobutyric acid, N-Methylglycine, sarcosine, 3-Aminoisobutyric acid, N-Methylisoleucine, 2-Aminopimelic acid, 6-N-Methyllysine, 2,4-Diaminobutyric acid, N-Methylvaline, Desmosine, Norvaline, 2,2'-Diaminopimelic acid, Norleucine, 2,3- Diaminopropionic acid, Ornithine, and N-Ethylglycine. The term“amino acid residue” may include L isomers or D isomers of any of the aforementioned amino acids.

[0070] Other examples of nonstandard, noncanonical, or unnatural amino acids include, but are not limited, to a p-acetyl-L-phenylalanine, a p-iodo-L-phenylalanine, an O-methyl-L-tyrosine, a p-propargyloxyphenylalanine, a p-propargyl-phenylalanine, an L- 3-(2-naphthyl)alanine, a 3 -methyl-phenylalanine, an O-4-allyl-L-tyrosine, a 4-propyl-L- tyrosine, a tri-0-acetyl-GlcNAcpb-serine, an L-Dopa, a fluorinated phenylalanine, an isopropyl-L-phenylalanine, a p-azido-L-phenylalanine, a p-acyl-L-phenylalanine, a p- benzoyl-L-phenylalanine, an L-phosphoserine, a phosphonoserine, a phosphonotyrosine, a p-bromophenylalanine, a p-amino-L-phenylalanine, an isopropyl-L-phenylalanine, an unnatural analogue of a tyrosine amino acid; an unnatural analogue of a glutamine amino acid; an unnatural analogue of a phenylalanine amino acid; an unnatural analogue of a serine amino acid; an unnatural analogue of a threonine amino acid; an unnatural analogue of a methionine amino acid; an unnatural analogue of a leucine amino acid; an unnatural analogue of a isoleucine amino acid; an alkyl, aryl, acyl, azido, cyano, halo, hydrazine, hydrazide, hydroxyl, alkenyl, alkynl, ether, thiol, sulfonyl, seleno, ester, thioacid, borate, boronate, l5ufal5hor, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, hydroxylamine, keto, or amino substituted amino acid, or a combination thereof; an amino acid with a photoactivatable cross-linker; a spin-labeled amino acid; a fluorescent amino acid; a metal binding amino acid; a metal-containing amino acid; a radioactive amino acid; a photocaged and/or photoisomerizable amino acid; a biotin or biotin-analogue containing amino acid; a keto containing amino acid; an amino acid comprising polyethylene glycol or polyether; a heavy atom substituted amino acid; a chemically cleavable or photocleavable amino acid; an amino acid with an elongated side chain; an amino acid containing a toxic group; a sugar substituted amino acid; a carbon-linked sugar-containing amino acid; a redox-active amino acid; an a-hydroxy containing acid; an amino thio acid; an a, a disubstituted amino acid; a b-amino acid; a g-amino acid, a cyclic amino acid other than proline or histidine, and an aromatic amino acid other than phenylalanine, tyrosine or tryptophan.

[0071] As used herein, a“peptide” is defined as a short polymer of amino acids, of a length typically of 20 or less amino acids, and more typically of a length of 12 or less amino acids (Garrett & Grisham, Biochemistry, 2^nd edition, 1999, Brooks/Cole, 110). In some embodiments, a peptide as contemplated herein may include no more than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. A polypeptide, also referred to as a protein, is typically of length > 100 amino acids (Garrett & Grisham, Biochemistry, 2^nd edition, 1999, Brooks/Cole, 110). A polypeptide, as contemplated herein, may comprise, but is not limited to, 100, 101, 102, 103, 104, 105, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about

200, about 210, about 220, about 230, about 240, about 250, about 275, about 300, about

325, about 350, about 375, about 400, about 425, about 450, about 475, about 500, about

525, about 550, about 575, about 600, about 625, about 650, about 675, about 700, about

725, about 750, about 775, about 800, about 825, about 850, about 875, about 900, about

925, about 950, about 975, about 1000, about 1100, about 1200, about 1300, about 1400, about 1500, about 1750, about 2000, about 2250, about 2500 or more amino acid residues.

[0072] A peptide as contemplated herein may be further modified to include non amino acid moieties. Modifications may include but are not limited to acylation ( e.g ., O- acylation (esters), N-acylation (amides), S-acylation (thioesters)), acetylation (e.g., the addition of an acetyl group, either at the N-terminus of the protein or at lysine residues), formylation lipoylation (e.g., attachment of a lipoate, a C8 functional group), myristoylation (e.g., attachment of myristate, a C14 saturated acid), palmitoylation (e.g., attachment of palmitate, a C16 saturated acid), alkylation (e.g., the addition of an alkyl group, such as an methyl at a lysine or arginine residue), isoprenylation or prenylation (e.g., the addition of an isoprenoid group such as famesol or geranylgeraniol), amidation at C-terminus, glycosylation (e.g., the addition of a glycosyl group to either asparagine, hydroxylysine, serine, or threonine, resulting in a glycoprotein). Distinct from glycation, which is regarded as a nonenzymatic attachment of sugars, polysialylation (e.g., the addition of polysialic acid), glypiation (e.g., glycosylphosphatidylinositol (GPI) anchor formation, hydroxylation, iodination (e.g., of thyroid hormones), and phosphorylation (e.g., the addition of a phosphate group, usually to serine, tyrosine, threonine or histidine).

[0073] As used herein, “translation template” refers to an RNA product of transcription from an expression template that can be used by ribosomes to synthesize polypeptides or proteins.

[0074] The term“reaction mixture,” as used herein, refers to a solution containing reagents necessary to carry out a given reaction. A reaction mixture is referred to as complete if it contains all reagents necessary to perform the reaction. Components for a reaction mixture may be stored separately in separate container, each containing one or more of the total components. Components may be packaged separately for commercialization and useful commercial kits may contain one or more of the reaction components for a reaction mixture.

[0075] The steps of the methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The steps may be repeated or reiterated any number of times to achieve a desired goal unless otherwise indicated herein or otherwise clearly contradicted by context.

[0076] Preferred aspects of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred aspects may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect a person having ordinary skill in the art to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above- described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0077] Cell-Free Protein Synthesis

[0078] Cell-free protein synthesis (CFPS) and methods for making cell extracts for use in CFPS are known in the art. (See, e.g., Carlson el al,“Cell-free protein synthesis: Applications come of age,” Biotech. Adv. Vol. 30, Issue 5, Sept-Oct 2012, Pages 1185- 1194; Hodgman el al. ,“Cell-free synthetic biology: Thinking outside the cell,” Metabol. Eng. Vol. 14, Issue 3, May 2012, Pages 261-269; and Harris et al,“Cell-free biology: exploiting the interface between synthetic biology and synthetic chemistry,” Curr. Op. Biotech. Vol. 23, Issue 5, October 2012, Pages 672-678; see also U.S. Patent Nos. 7,312,049; 7,008,651; and 6994986; see also U.S. Published Application Nos. 20170306320; 20160362708; 20160060301; 20120088269; 20090042244; 2008024821; 20080138857; 20070154983; 20070141661; 20050186655; 200501480461 20050064592; 20050032086; 20040209321; and 20040038332; the contents of which are incorporated herein by reference in their entireties).

[0079] The disclosed compositions may include platforms for preparing a sequence defined biopolymer of protein in vitro. The platforms for preparing a sequence defined polymer or protein in vitro comprises an extract from an organism, and in particular a species of plant, such as an extract from chloroplasts of a plant, such as Nicotiana tabacum.

[0080] Because CFPS exploits an ensemble of catalytic proteins prepared from the crude lysate of cells, the cell extract (whose composition is sensitive to growth media, lysis method, and processing conditions) is an important component of extract-based CFPS reactions. A variety of methods exist for preparing an extract competent for cell- free protein synthesis, including those disclosed in U.S. Published Application No. 20140295492, published on Oct. 2, 2014, which is incorporated by reference. [0081] The platform may comprise an expression template, a translation template, or both an expression template and a translation template. The expression template serves as a substrate for transcribing at least one RNA that can be translated into a sequence defined biopolymer (e.g., a polypeptide or protein). The translation template is an RNA product that can be used by ribosomes to synthesize the sequence defined biopolymer. In certain embodiments the platform comprises both the expression template and the translation template. In certain specific embodiments, the platform may be a coupled transcription/translation ("Tx/Tl") system where synthesis of translation template and a sequence defined biopolymer from the same cellular extract.

[0082] The platform may comprise one or more polymerases capable of generating a translation template from an expression template. The polymerase may be supplied exogenously or may be supplied from the organism used to prepare the extract. In certain specific embodiments, the polymerase is expressed from a plasmid present in the organism used to prepare the extract and/or an integration site in the genome of the organism used to prepare the extract.

[0083] The platform may comprise an orthogonal translation system. An orthogonal translation system may comprise one or more orthogonal components that are designed to operate parallel to and/or independent of the organism's orthogonal translation machinery. In certain embodiments, the orthogonal translation system and/or orthogonal components are configured to incorporation of unnatural amino acids. An orthogonal component may be an orthogonal protein or an orthogonal RNA. In certain embodiments, an orthogonal protein may be an orthogonal synthetase. In certain embodiments, the orthogonal RNA may be an orthogonal tRNA or an orthogonal rRNA. An example of an orthogonal rRNA component has been described in U.S. Published Application Nos. 20170073381 and 20160060301, the contents of which are incorporated by reference in their entireties. In certain embodiments, one or more orthogonal components may be prepared in vivo or in vitro by the expression of an oligonucleotide template. The one or more orthogonal components may be expressed from a plasmid present in the genomically recoded organism, expressed from an integration site in the genome of the genetically recoded organism, co-expressed from both a plasmid present in the genomically recoded organism and an integration site in the genome of the genetically recoded organism, express in the in vitro transcription and translation reaction, or added exogenously as a factor (e.g., a orthogonal tRNA or an orthogonal synthetase added to the platform or a reaction mixture.

[0084] Platforms Comprising Extracts from Plant Chloroplasts

[0085] The disclosed compositions (or systems) may include platforms for preparing a sequence defined biopolymer or protein in vitro, where the platform comprising an extract prepared from plant chloroplast, in particular, plant chloroplasts from Nicotiana tabacum and maize.

[0086] The platforms disclosed herein may include additional components, for example, one or more components for performing CFPS. Components may include, but are not limited to amino acids which optionally may include noncanonical amino acids, NTPs, salts (e.g., sodium salts, potassium salts, and/or magnesium salts), cofactors (e.g., nicotinamide adenine dinucleotide (NAD) and/or coenzyme- A (Co A)), an energy source and optionally an energy source comprising a phosphate group (e.g., phosphoenol pyruvate (PEP), ATP, or creatine phosphate), a translation template (e.g., a non-native mRNA that is translated in the platform) and/or a transcription template (e.g., a template DNA for synthesizing a non-native mRNA that is translated in the platform), and any combination thereof.

[0087] In some embodiments, the platform may comprise an energy source and optionally an energy source comprising a phosphate group (e.g., phosphoenol pyruvate (PEP), ATP, or creatine phosphate), where the energy source is present in the platform at a concentration of greater than about 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, or 90 mM (preferably greater than about 67 mM), but less than about 100 mM, or within a concentration range bounded by of these values. [0088] In some embodiments, the platform further comprises a source of potassium (K⁺)(such as a potassium salt such as potassium glutamate), where the platform comprises potassium at a concentration greater than about 50 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, or 450 mM (preferably about 300 mM), but less than about 500 mM, or within a concentration range bounded by of these values,

[0089] The disclosed platforms and cell extracts may be utilized in methods for preparing a sequence defined biopolymer or protein in vitro. The disclosed methods typically include translating in vitro a translation template (e.g., mRNA) encoding the sequence defined biopolymer or protein in the platform of any of the foregoing claims. Optionally, the disclosed methods may include transcribing a transcription template (e.g., DNA) in the platform to provide the translation template.

[0090] The disclosed methods may be performed under conditions that are suitable for extracts prepared from plant chloroplasts. In some embodiments, the disclosed methods are performed at a temperature between about 20-40 °C, and preferably at a temperature of about 30 °C.

[0091] The disclosed methods may be performed to synthesize any sequence defined biopolymer or protein. In some embodiments, the sequence defined polymer or protein is a therapeutic protein and/or the method may utilized to identify therapeutic proteins or biomaterials by translating a library of transcription templates. In some embodiments, the disclosed methods may be performed to optimize in vitro translation conditions for a cellular extract prepared from a species of plant.

[0092] Kits also are contemplated herein. In some embodiments, the contemplated kits comprise as components: (a) a cellular extract prepared from plant chloroplasts; and (b) a reaction mixture for transcribing and/or translating an mRNA. Suitable components for the reaction mixture of the disclosed kits may include, but are not limited to, amino acids which optionally may include noncanonical amino acids, NTPs, salts (e.g., sodium salts, potassium salts, and/or magnesium salts), cofactors (e.g., nicotinamide adenine dinucleotide (NAD) and/or coenzyme-A (CoA)), an energy source and optionally an energy source comprising a phosphate group (e.g., ATP or creatine phosphate).

ILLUSTRATIVE EMBODIMENTS

[0093] The following Embodiments are illustrative and should not be interpreted to limit the scope of the claimed subject matter.

[0094] 1. A cell-free protein synthesis platform for in vitro transcription of mRNA and/or translation of polypeptides, the platform comprising as a component: (a) a chloroplast extract prepared from plants, optionally plants that are grown for about 4-8 weeks, optionally wherein the plants are grown in about 6 hours dark and in about 18 hours light during the 4-8 weeks, and optionally incubating the plants in total dark for 24 hours, preferably 10-12 hours before preparing the chloroplast extract; and the platform optionally comprising one or more of the following components: (b) a reaction buffer; (c) an RNA polymerase; and (d) the transcription template, wherein the RNA polymerase is capable of transcribing the transcription template to form a translation template and the chloroplast extract can sustain protein synthesis through a combined transcription/translation reaction.

[0095] 2. The platform of claim 1, wherein the chloroplast extract is prepared from a species selected from Nicotiana spp. (e.g., tobacco plant).

[0096] 3. The platform of embodiment 1, wherein the chloroplast extract is prepared from a species selected from Maize spp (e.g, com plant), Glycine spp. (e.g. soybean plant), Oryza spp. (e.g, rice plant), and Triticum spp. (e.g, wheat plant).

[0097] 4. The platform of embodiment 1, wherein the chloroplast extract is prepared from a species selected from Solanum spp. (e.g., Solanum lycopersicum (tomato plant) or Solanum tubersosum (potato plant)).

[0098] 5. The platform of any of embodiments 1-4, wherein the chloroplast extract is prepared from a plant that is engineered to be deficient in a negative effector for cell-free protein synthesis (CFPS), or from a plant that is engineered to be deficient in a negative effector for combined transcription and translation.

[0099] 6. The platform of any of the foregoing embodiments, wherein the chloroplast extract is prepared from a plant that has been engineered to express T7 RNA polymerase in the chloroplast or in the nucleus and T7 RNA polymerase is targeted to the chloroplast.

[00100] 7. The platform of any of the foregoing embodiments, wherein the platform or any component of the platform (e.g., the chloroplast extract) is preserved, such as for example through freeze-drying.

[00101] 8. A method for in vitro transcription of mRNA and/or translation of mRNA to prepare a polypeptide, the method comprising reacting a reaction mixture comprising: (a) an extract from chloroplasts of plants, wherein the plants optionally are grown for about 4-8 weeks and preferably for about 6 weeks; (b) a template for transcription of the mRNA (e.g., a DNA encoding the mRNA which may be linear or circular such as a plasmid) and/or a template for translation to prepare the polypeptide (e.g., an mRNA); (c) monomers for synthesis of the mRNA (e.g., one or more of ATP, CTP, GTP, and/or UTP) and/or the polypeptide (e.g., one or more of any of the twenty (20) canonical amino acids or non-canonical amino acids); (d) co-factors, enzymes and/or other reagents necessary for the transcription and/or translation; and (e) magnesium at a concentration of from about 5 mM to about 20 mM, optionally wherein the method is performed using an automated system and/or mechanized system which optionally comprises a liquid-handling robot, for example, to assemble the reaction mixture.

[00102] 9. The method of embodiment 8, wherein the concentration of amino acids (as a group or individually) is between about 0.5 to 4 mM, and preferably about 2 mM. [00103] 10. The method of embodiment 8 or 9, wherein the reaction mixture further comprises potassium at a concentration of about 0-200 mM, and preferably about 100 mM.

[00104] 11. The method of any of embodiments 8-10, wherein the reaction mixture further comprises salts at a total concentration of between about 50-400 mM.

[00105] 12. The method of any of embodiments 8-11, wherein the reaction mixture further comprises an energy regeneration system that comprises creatine kinase and creatine phosphate.

[00106] 13. The method of any of embodiments 8-12, wherein the reaction mixture further comprises at least one macromolecular crowding agent (e.g., polyethylene glycol or Ficol).

[00107] 14. The method of any of embodiments 8-13, wherein the chloroplast extract is prepared by a method that includes a step of adding glycerol to the chloroplast prior to lysing the chloroplasts (e.g., lysis by freezing), preferably a step of adding glycerol at a concentration of about 5-15% or about 10% to the chloroplasts prior to lysing the chloroplasts.

[00108] 15. The method of any of embodiments 8-14, wherein the template for the mRNA includes modifications in the 3’UTR that facilitate efficient transcription and/or translation.

[00109] 16. The method of any of embodiments 8-15, wherein the template for the mRNA includes modifications in the 5’UTR that facilitate efficient transcription and/or translation.

[00110] 17. The method of any of embodiments 8-16, wherein the reaction mixture further comprises a DNA-dependent RNA polymerase.

[00111] 18. The method of embodiment 17, wherein the DNA-dependent RNA polymerase is a bacteriophage DNA-dependent RNA, such as T7 RNA polymerase. [00112] 19. The method of any of embodiments 8-18, wherein the method is performed as a batch reaction.

[00113] 20. The method of any of embodiments 8-18, wherein the method is performed as a semi-continuous reaction.

[00114] 21. The method of any of embodiments 8-18, wherein the method is performed as a semi-continuous reaction.

[00115] 22. The method of any of embodiments 8-21, wherein the method is performed at a temperature between about 20-40 °C.

[00116] 23. The method of any of embodiments 8-22, wherein the reaction mixture or any component of the reaction mixture (e.g., the extract from chloroplasts) is preserved, such as for example through freeze-drying.

[00117] 24. The method of any of embodiments 8-23, wherein an RNAse inhibitor is added to the reaction mixture to enhance

[00118] 25. A method comprising: (a) creating a test library of genetic parts of plants (e.g., one or more of test promoters, test terminators, test ribosome binding sites, and the like); and (b) testing the function of the genetic parts of the test library in a platform comprising: (i) a cellular extract prepared from a plant chloroplast; and (ii) a reaction mixture for transcribing and/or translating an mRNA; and (c) optionally assessing gene expression, and optionally using information obtained from assessing gene expression to modify gene expression in a plant (e.g., by creating a genetically modified plant); and (d) optionally wherein the method is used to is used to characterize and assess the genetic parts and fine-tune gene expression prior to studying the genetic parts and gene expression in plants.

[00119] 26. A method comprising: (a) creating a test library of codon-optimized constructs of a gene product; and (b) testing expression of the codon-optimized constructs of the test library in a platform comprising: (i) a cellular extract prepared from a plant chloroplast; and (ii) a reaction mixture for transcribing and/or translating an mRNA; and (c) optionally using information obtained from testing expression of the codon-optimized constructs to modify expression in a plant (e.g., by creating a genetically modified plant); and (d) optionally wherein the method is used to is used to characterize and assess codon optimization and fine-tune of gene expression prior to studying codon optimization and fine-tuning of gene expression in plants.

[00120] 27. A method comprising: (a) creating a test library of constructs expressing several genes; and (b) testing expression of the constructs of the test library in a platform comprising: (i) a cellular extract prepared from a plant chloroplast; and (ii) a reaction mixture for transcribing and/or translating an mRNA; and (c) optionally using information obtained from testing expression of the construct to modify gene expression in a plant (e.g., by creating a genetically modified plant); and (d) optionally wherein the method is used to is used to provide information to a geneticist regarding the design of multi-gene functions.

[00121] 28. A method comprising: (a) creating a test library of constructs of a biosynthetic pathway expressing enzymes; and (b) testing the constructs of the test library in a platform comprising: (i) a cellular extract prepared from a plant chloroplast; and (ii) a reaction mixture for transcribing and/or translating an mRNA; and (c) assessing gene expression, metabolite production, requirement of accessory proteins or other molecules, and/or metabolic parameters; (d) optionally using information obtained from testing the constructs to modify gene expression in a plant (e.g., by creating a genetically modified plant); and (e) optionally wherein the method is used to is used to provide information to a geneticist regarding the design and function of enzyme pathways for example in order to fine-tune expression levels of the enzymes and testing possible accessory proteins or other molecules required for proper pathway function.

[00122] 29. A method comprising: (a) creating plant specific sensors comprising genetic circuits that respond to external commands; and (b) testing the sensors in a platform comprising: (i) a cellular extract prepared from a plant chloroplast; and (ii) a reaction mixture for transcribing and/or translating an mRNA; and (c) assessing sensor response and circuit behavior; and (d) optionally wherein the method is used to is used to provide information to a geneticist regarding the design and function of sensor and genetic circuits.

[00123] 30. A kit comprising as components: (a) a cellular extract prepared from a plant chloroplast (optionally wherein the cellular extract is preserved, such as for example by freeze-drying); and (b) a reaction mixture for transcribing an mRNA from a DNA template and or for translating an mRNA to prepare a polypeptide (optionally wherein the reaction mixture is preserved, such as for example by freeze-drying).

[00124] 31. The kit of embodiment 30, wherein the plant is Nicotiana tabacum.

[00125] 32. The kit of embodiment 30 or 31, wherein the reaction mixture comprises one or more components selected from the group consisting of amino acids which optionally may include non-canonical amino acids, NTPs, salts, cofactors, an energy source and optionally an energy source comprising a phosphate group.

[00126] 33. A method for preparing an extract from chloroplasts of a plant, the method comprising: (a) obtaining a plant optionally grown for about 4-8 weeks and preferably about 6 weeks; (b) isolating the chloroplasts from the plant; (c) adding a polyol compound (e.g., glycerol) to the isolated chloroplasts (e.g., at a concentration of about 5- 15% and preferably at a concentration of about 10%); and (d) lysing the chloroplasts (e.g., by freezing) and separating the extract from the lysed chloroplasts (e.g., separating a soluble extract from the lysed chloroplasts).

EXAMPLES

[00127] The following Examples are illustrative and should not be interpreted to limit the scope of the claimed subject matter.

[00128] Example 1 - A Novel Combined Transcription and Translation Platform

Derived From Plant Chloroplasts [00129] Abstract

[00130] The current state of the art in transformation of plant chloroplasts is a laborious, low-throughput method that takes approximately one year to produce a stably transgenic plant. As a result, developments in plant biotechnology and our understanding of the basic biology of plants have lagged far behind what we have been able to achieve with mammalian or prokaryotic biology, in spite of the fact that plants are considered to be the source of a rich potential for pharmaceutically and technologically relevant natural products. Here we present a chloroplast cell-free protein synthesis platform derived from Nicotiana tabacum that would allow for high-throughput screening of genetic parts and allow for targeted design before plant transformation. This is the first ever - to our knowledge - combined transcription and translation system from plant chloroplasts. We have developed a series of protocols comprising plant growth conditions, a harvest and lysis protocol, and optimized CFPS reaction mixture that can be used to generate chloroplast extract for prototyping genetic parts. The optimized system is capable of producing up to ~30pg/mL of luciferase reporter protein, which can be detected by plate reader and affords enough dynamic range to begin to determine nuances in genetic parts libraries.

[00131] Applications

[00132] Applications for the disclosed technology include, but are not limited to: (i)

In vitro screening of enzyme pathway variants for plant metabolic engineering; (ii) Pre screening DNA designs with chloroplast CFPS allows for a dramatic reduction in iteration time for research and development; (iii) Synthetic biology applications; (iv) Fundamental studies of the chloroplast translation apparatus and harnessing translation machinery for novel functions; (v) High-throughput interrogation and characterization of organismal genetic parts (promoters, terminators, ribosome binding sites, etc.); and (vi) Agriculture and Crop Science. [00133] Advantages

[00134] Applications for the disclosed technology include but are not limited to the following aspects.

[00135] The current state of the art in chloroplast transformation takes approximately one year to produce transformants, meaning that design iterations occur annually. Chloroplast transformation is a laborious process that involves bombardment of plant tissue with gold particles loaded with DNA. This method is not sufficiently high- throughput for prototyping genetic parts. The disclosed technology can be utilized to significantly curtail the amount of time needed to perform multiple genetic tests. For example, instead of performing 10 genetic tests in 9 month, one could perform 10,000 tests in a week.

[00136] While it is possible to use plant cell culture to study candidates for transformation into plants and some plant cell lines such as BY-2 cell lines contain chloroplasts, their chloroplasts are undifferentiated, and therefore less suitable to mimic cellular processes of a fully developed functional chloroplast. . Similarly, wheat germ extract could be used to prototype nuclear modifications. However, the chloroplast is a more desirable location to stably express exogenous DNA because there is no silencing of organelle DNA. In addition, several genes can be expressed from an operon and the maternal inheritance of this subcellular compartment prevents exogenous gene escape through pollen.

[00137] Brief Summary of Technology

[00138] Cell-free protein synthesis (CFPS) is fueling numerous applications as a powerful in vitro expression system. Yet, there is little available for in vitro genetic prototyping for plant biologists. We have applied our expertise in cell-free systems to the existing work on in vitro translation to develop the first ever combined transcription- translation cell free platform derived from chloroplasts. We chose Nicotiana tabacum for its large leaf size and rapid growth, as well as well as the fact that it grows readily in an indoor setting. Currently, we have demonstrated the ability to transcribe plasmid DNA in tobacco chloroplast cell-free extracts. Then, we next achieved combined transcription and translation by improving the plants’ growth conditions and selecting the BY genetic background. Next, we sought to improve yields by optimizing our harvest conditions. Initially, we collected leaves at 4 weeks post-germination, the earliest reasonable time based on tissue mass available from the plants. As a follow-up, we allowed the plants to recover for two weeks and harvested leaves again at 6 weeks. We found that chloroplast extract from 6-week-old plants is over six-fold more productive in CFPS than that from 4- week-old plants. Once we determined the optimal growth conditions, we sought to improve our harvest and lysis protocol. Surprisingly, we found that freezing chloroplasts resuspended in lysis buffer with 10% glycerol had a dramatically positive effect (which was surprising since this has not been done before). Specifically, results show that addition of glycerol prior to freezing improves yields 30-fold. Finally, we sought to optimize our cell-free reactions in a two-pronged approach. First we surveyed a number of plasmids with different 5’-UTR ad 3’-UTR to boost yields and validate our hypothesis that the chloroplast cell-free system could be used to assess and rank different genetic parts. Further, results from this library have shown that the 5’-UTR has shown to be more important than the 3’-UTR for high yields. Next, we optimized the reaction environment by varying the levels of small molecules, enzymes, and crowding in our reactions. We found that high concentrations of amino acids were important to obtain high yields. Currently our batch reactions produce stable, active luciferase or GFP for about three hours and can be used to rank 5’-UTRs. We expect that this technology will be highly desirable to large biotechnology companies that seek to prototype on a more rapid timescale.

[00139] Problems Solved

[00140] Problems solved by the disclosed technology include but are not limited to the following aspects. Technological developments in plant biology have historically been slow, in spite of the fact that our early understanding of genetics was pioneered in plants. Previously, prototyping in BY -2 cell culture has informed transformation into the nuclear genome, though this is not necessarily a high-throughput process and nuclear transformation is susceptible to gene silencing. Transformation into the chloroplast is more generationally stable, but is highly time-consuming, requiring one year to produce transformants. There is also no known way of pre-screening candidates for transformation in a chloroplast-like environment. As a result, the current compromise is to devote a staggering number of person-hours to development of a small number of candidates or to prototype in a nuclear context.

[00141] Commercial Aspects

[00142] Commercial aspects of the disclosed technology include but are not limited to the following. Previous researchers have sought to produce chloroplast extracts for in vitro translation, but this has proved challenging, likely due to the fact that freeze/thaw and handling can rupture chloroplasts. Our method includes addition of 10% glycerol lysis buffer prior to freezing, which ensures active extract. It is at present unclear if this prevents lysis entirely or merely protects chloroplast proteins and structures through the thawing process.

[00143] Plant biotechnology companies seek to increase their design-build-test cycles to increase revenue. Our platform could be used to screen thousands of genetic parts in the amount of time that it would take to produce one round of chloroplast transformants. Currently, the system is able to produce up to 28 pg/mL of active luciferase, which we have shown to be enough dynamic range to rank a small library of 5’-UTR and 3’-UTR variants. This technology could be combined with the well- understood practice of tobacco chloroplast transformation to produce large amounts of medically or agriculturally relevant compounds. Alternatively, our methods could be applied to chloroplasts derived from therapeutically or agriculturally relevant species to better prototype in more distantly related plant species such as grasses, trees, or algae. Our initial discovery has opened up the possibility to manipulate levels of natural products in many different areas of plant biotechnology. [00144] This system is the first ever chloroplast-derived CFPS system capable of transcription and translation and is more high-yielding than previously reported translation-only systems. While other plant-derived systems have been published (wheat germ extract and BY-2 extract), these systems are not suited to prototyping for the chloroplast, which is a more stable transformation cite than the nucleus. This system will enable technological discovery by generating a method for rapid prototyping in a plant context.

[00145] Results

[00146] Aspects of this Example are further described in the figures that accompany this application and the corresponding figure legends.

[00147] Figure 1 provides an overview of the isolation of intact chloroplasts from plant material and preparation of an extract from the isolated chloroplasts for use in cell- free transcription and/or translation. In a first step, plant material, such as leaf material is ground and chloroplasts are separated from the ground plant material via low speed centrifugation (e.g., 4000 x g). Next, the separated chloroplasts are overlaid on a gradient and subjected to higher speed centrifugation (10000 x g) to separate broken chloroplasts from intact chloroplasts. Intact chloroplasts are isolated and subjected to lysis (e.g., via freezing) to prepare an extract which may be utilized as part of a cell-free protein synthesis (CFPS) reaction mixture comprising the extract, a DNA expression construct, and additional additives for protein synthesis.

[00148] The activity of a lysate in cell-free protein synthesis reactions is correlated with the amount of intact chloroplasts utilized to prepare the lysate. Figure 2 illustrates the relative protein concentration of ribulose- 1 ,5-bisphosphate carboxylase/oxygenase (Rubisco), an enzyme present in chloroplasts, versus the concentration of membrane proteins in a extract can be utilized as an indicator of intact chloroplasts versus broken chloroplasts in plant material. [00149] Chloroplast extracts can be utilized in cell-free systems to express mRNA.

Figure 3 illustrates the production of mRNA in a cell-free system comprising a chloroplast extract as a function of the concentration of a DNA template for mRNA present in the system. As illustrated, mRNA production (concentration) was correlated with the concentration of DNA template added to the cell-free system.

[00150] Chloroplast extracts also can be utilized to express a protein. Figure 4 illustrates the results of experiments in which a template DNA encoding a fluorescent target protein was transcribed and translated in cell-free systems comprising a chloroplast extract. Protein production was only observed in the presence of template DNA.

[00151] Having established a strategy to prepare chloroplast extracts that were competent for transcription and translation, we set out to improve protein yields in our cell-free protein synthesis (CFPS) systems to ~ 1-5 pg/mL.

[00152] First, because divalent cations are known to be required in CFPS systems, we assessed protein production in our chloroplast extract CFPS system in the presence of the divalent cation Mg²⁺. Figure 5 illustrates that divalent cations such as Mg²⁺ are required for efficient protein production in a cell-free system comprising a chloroplast extract in which a template DNA encoding a fluorescent target protein was transcribed and translated.

[00153] We also assessed whether we could obtain higher efficiency of protein production in our CFPS system by using extracts prepared from plants that had been grown for different periods of time. Figure 6 illustrates that chloroplast extracts prepared from chloroplasts isolated from plants that were grown at six (6) weeks versus four (4) weeks exhibit better performance regarding protein production in a cell-free free system. Chloroplasts from 6 week old plants are over six-fold more productive in CFPS than those from 4 week old plants

[00154] Because exposure to light is known to increase the production of starch in chloroplasts which may make chloroplasts more susceptible to membrane breakage during isolation, we assessed whether we could obtain extracts that exhibited higher production in our CFPG system by using extracts prepared from plants that had been exposed to darkness prior to our isolating chloroplasts from the plants and preparing extracts. Figure 7 illustrates a method to increase lysate activity related to total protein content by dark incubation of plants before extract preparation. After 6-24 h of dark incubation starch granules are reduced in the chloroplasts as observed by light microscopic images which results higher total protein content as measured with Bradford assay and lysate productivity as measured by expressed luciferase in the chloroplast cell-free system. Figure 8 illustrates the results of semi-continuous and batch reaction of extracts from light/dark incubated plants. Semi-continuous reaction conditions were observed to increase protein output independent of light/dark treatment, although substrate replenishment appeared different in "light" versus "dark" extracts. This might be the result of higher activity of proteases in dark incubated plants or that dark exposure might lead to different metabolic/translational states. Dark and light exposed chloroplast extracts could have different applications.

[00155] Because lysing may disrupt some of the supramolecular structures of components utilized in transcription/translation, we tested whether we could add an agent that may stabilize supramolecular structures after lysis. As such, we assessed whether the addition of a polyol such as glycerol to our isolated chloroplasts prior to lysis by freezing could improve productivity of the lysates. As illustrated in Figure 9, protein production is improved in our chloroplast CFPS system when a polyol such as glycerol is added, and addition of glycerol prior to lysis by freezing the chloroplasts improved yields by 30-fold.

[00156] We also assessed whether we could obtain additive improvement in protein productivity by utilizing all of the individual conditions that were observed to improve protein in combination. Figure 10 illustrates the results of combined optimization on protein production in a cell-free system comprising a chloroplast extract, including addition of a polyol, timing of growth in plants from which the chloroplast extract is prepared, codon optimization for expression in the plants from which the chloroplast extract is prepared. [00157] We next assessed whether we could modify elements that are present in the 5'-UTR and 3'-UTR of our DNA expression template in order to modulate protein production. Figure 11 illustrates that protein expression is influenced by modifications in the 5'-UTR, such as modifications in the ribosome binding site (RBSD) and in the 3'-UTR, such as modifications in the transcription terminator.

[00158] We next tested various salt, buffer, and pH conditions to determine whether we could optimize yields in our chloroplast extract CFPS system. Figure 12 illustrates the effect of magnesium acetate versus magnesium glutamate on protein production in a cell- free system comprising a chloroplast extract. Magnesium acetate at a concentration between about 6-11 mM (e.g., about 8-10 mM) was observed to optimize protein production.

[00159] Figure 13 illustrates the effect of adding potassium on protein production in a cell-free system comprising a chloroplast extract. A concentration of potassium of about 50-80 mM (e.g., about 55-70 mM) was observed to optimize protein production.

[00160] Figure 14 illustrates the effect of pH in the presence of a HEPES buffered system versus a Tris buffered system on protein production in a cell-free system comprising a chloroplast extract. HEPES was observed to perform better than Tris in optimizing protein production and a pH of less than about 7.8 was observed to be optimal for protein production (e.g., less than about 7.7, 7.6, or 7.5 and/or higher than about 6.8).

[00161] Figure 15 illustrates the effect of adding ammonium on protein production in a cell-free system comprising a chloroplast extract. Protein production was optimal when ammonium was present at a concentration of about 20-60 mM (e.g., about 30-50 mM).

[00162] Figure 16 illustrates the effect of amino acid concentration on protein production in a cell-free system comprising a chloroplast extract. Protein production was optimal when amino acids were present at a concentration of about 1-3 mM (e.g., about 2 mM). [00163] Figure 17 illustrates the effect of HEPES concentration at a pH of 7.3 on protein production in a cell-free system comprising a chloroplast extract. Protein production was optimal when HEPES was present at a concentration of about 20-50 mM.

[00164] In summary, we observed that optimizing the pH and the concentration of ammonium, HEPES, and amino acids, we could increase the protein yield of an extract from ~7 pg/mL to 30 pg/mL.

[00165] We also assessed which energy regeneration systems resulted in highest efficiency in our chloroplast extract CFPS system. Figure 18 illustrates the effect of different energy regeneration systems on protein production in a cell-free system comprising a chloroplast extract, including a phosphoenolpyruvate (PEP) system, a PEP/ pyruvate kinase (PyK) system, and a creatine phosphate (CP)/ creatine kinase (CK) system. Protein production required the CP/CK system. Figure 19 illustrates the effect of different concentrations of a creatine phosphate (CP)/ creatine kinase (CK) energy regeneration system on protein production in a cell-free system comprising a chloroplast extract. A concentration of about 0.27-0.33 mg/mL was observed to optimize protein production.

[00166] We also assessed whether we could improve protein production by optimizing the concentration of the DNA-dependent RNA polymerase from T7 (T7 RNAP) in our chloroplast extract CFPS system. Figure 20 illustrates the effect of different concentration of T7 RNAP on protein production in a cell-free system comprising a chloroplast extract.

[00167] We also assessed whether we could improve protein production by optimizing the concentration of DNA template in our chloroplast extract CFPS system. Figure 21 illustrates the effect of final DNA template concentration at two different concentrations of T7 polymerase on protein production in a cell-free system comprising a chloroplast extract. [00168] We also assessed whether we could improve protein production by adding macromolecular crowding agents to our chloroplast extract CFPS system. Figure 22 illustrates the effect of different macromolecular crowding agents on protein production in a cell-free system comprising a chloroplast extract, including Ficoll 400, PEG 600, PEG 3350, and FEG 8000. Figure 23 illustrates the effect of different macromolecular crowding agents at different concentrations on protein production in a cell-free system comprising a chloroplast extract, including Ficoll 400, PEG 3350, and PEG 8000 at one of 0.50%, 1%, 2%, or 4%.

[00169] We also assessed how the concentration of chloroplast extract could influence protein production in our chloroplast extract CFPS system. Figure 24 illustrates the effect of chloroplast extract concentration on protein production in a cell-free system comprising the chloroplast extract. Protein production was optimal at an extract concentration of between about 40-70% (e.g., about 50-60%).

[00170] We also assessed protein production versus time. Figure 25 illustrates protein production versus time in two different cell-free systems comprising a chloroplast extract. We observed that protein production increased from time 0-3 hours and achieved a steady state afterward.

[00171] We also assessed whether we could improve protein production by inhibiting mRNA degradation. Figure 26 shows the effect of RNAse inhibitor on chloroplast cell-free reaction. 0, 0.5 and 1 U/pL RNAse inhibitor was added. The results demonstrate that 0.5 U/pL RNAse inhibitor enhances protein production of the cell-free system, but that 1 U/pL RNAse inhibitor has no additional effect.

[00172] We also assessed whether we could utilize linear DNA templates for protein production. Figure 27 illustrates that linear templates or plasmid DNA can be used in chloroplast cell free reactions to produce protein, with linear templates offering a slight boost to yields and a faster turnaround time than plasmids. Linear templates prepared by PCR, then purified with a Qiagen cleanup kit (LT by EM) work as well as plasmid DNA. Also, DNA prepared with a Qiagen or ZymoPCR cleanup kit work equally well as a plasmid DNA as a DNA template for protein production.

[00173] Finally, we compared mRNA expression dynamics in our chloroplast extract CFPS system versus an E. coli extract CFPS system. Figure 28 illustrates different mRNA expression dynamics in chloroplast and E. coli cell-free systems. A construct (pY7lmRFPlSpA) expressing a fluorescent protein (mRFP) and a fluorescent RNA aptamer (spinach) was assayed in both chloroplast and E. coli cell-free systems. The results in Figure 28 show that mRNA levels are stable in the chloroplast cell-free system during 8 hours (or more).

[00174] Example 3 - Methods of In Vitro Protein Synthesis and Prototyping of

Genetic Expression in Plants Utilizing Cell-Free Protein Synthesis Platforms Comprising

Chloroplast Extracts

[00175] Abstract

[00176] Chloroplasts are attractive targets of plant engineering with high foreign protein production capacity and genetic control that enables the expression of several genes stacked into operons. Transferring multi-genic functions requires the characterization of genetic parts and fine-tuning of protein expression, but the time- consuming transformation of chloroplasts prevents engineers from carrying out this task in plants. To overcome this problem, a cell-free system - based on purified and subsequently lysed tobacco chloroplasts - was used which enables the expression of proteins from a template. This can be used to assess the impact of ribosome binding sites, terminators, promoters, and other genetic parts, as well as codon optimization on gene expression prior to testing in a host plant. The chloroplast cell-free system prevents mistakes like using wrong codon-optimization or ribosome binding sites, and could allow for 10,000s or more genetic constructs to be prototyped weekly. Those producing the desired expression pattern could be selected for plastid transformation in the plant. In addition, the cell-free system could also serve as a test-bed to finely balance expression of multi-gene clusters. \

[00177] Applications

[00178] Applications for the disclosed technology include, but are not limited to: (i)

In vitro screening of enzyme pathway variants for plant metabolic engineering; (ii) Pre screening DNA designs with chloroplast CFPS allows for a dramatic reduction in iteration time for research and development; (iii) Synthetic biology applications; (iv) Fundamental studies of the chloroplast translation apparatus and harnessing translation machinery for novel functions; (v) High-throughput interrogation and characterization of organismal genetic parts (promoters, terminators, ribosome binding sites, etc.); and (vi) Agriculture and Crop Science.

[00179] Advantages

[00180] Applications for the disclosed technology include but are not limited to the following aspects.

[00181] The current state of the art in chloroplast transformation takes approximately one year to produce transformants, meaning that design iterations occur annually. Chloroplast transformation is a laborious process that involves bombardment of plant tissue with gold particles loaded with DNA. This method is not sufficiently high- throughput for prototyping genetic parts. The disclosed technology can be utilized to significantly curtail the amount of time needed to perform multiple genetic tests. For example, instead of performing 10 genetic tests in 9 month, one could perform 10,000 tests in a week.

[00182] While it is possible to use plant cell culture to study candidates for transformation into plants and some plant cell lines such as BY-2 cell lines contain chloroplasts, their chloroplasts are undifferentiated, and therefore less suitable to mimic cellular processes of a fully developed functional chloroplast. . Similarly, wheat germ extract could be used to prototype nuclear modifications. However, the chloroplast is a more desirable location to stably express exogenous DNA because there is no silencing of organelle DNA. In addition, several genes can be expressed from an operon and the maternal inheritance of this subcellular compartment prevents exogenous gene escape through pollen.

[00183] Brief Summary of Technology

[00184] Cell-free protein synthesis (CFPS) is fueling numerous applications as a powerful in vitro expression system. Yet, there is little available for in vitro genetic prototyping for plant biologists. We have applied our expertise in cell-free systems to improve and show utility of a CFPS from chloroplasts. Currently, we have demonstrated a powerful approach for prototyping genetic parts in cell-free systems. The technology is further described in Figures 1-5. We expect that this technology will be highly desirable to large biotechnology companies that seek to prototype on a more rapid timescale.

[00185] Problems Solved

[00186] Problems solved by the disclosed technology include but are not limited to the following aspects. Technological developments in plant biology have historically been slow, in spite of the fact that our early understanding of genetics was pioneered in plants. Previously, prototyping in BY -2 cell culture has informed transformation into the nuclear genome, though this is not necessarily a high-throughput process and nuclear transformation is susceptible to gene silencing. Transformation into the chloroplast is more generationally stable, but is highly time-consuming, requiring one year to produce transformants. There is also no known way of pre-screening candidates for transformation in a chloroplast-like environment. As a result, the current compromise is to devote a staggering number of person-hours to development of a small number of candidates or to prototype in a nuclear context.

[00187] Commercial Aspects

[00188] Commercial aspects of the disclosed technology include but are not limited to the following. Chloroplasts are attractive targets of plant engineering with high foreign protein production capacity and genetic control that enables the expression of several genes stacked into operons. Transferring multi-genic functions requires the characterization of genetic parts and fine-tuning of protein expression, but the time- consuming transformation of chloroplasts prevents engineers from carrying out this task in plants. To overcome this problem, a cell-free system - based on purified and subsequently lysed tobacco chloroplasts - was used which enables the expression of proteins from a template. This technology could be combined with the well-understood practice of tobacco chloroplast transformation to produce large amounts of medically or agriculturally relevant compounds. Alternatively, our methods could be applied to chloroplasts derived from therapeutically or agriculturally relevant species to better prototype in more distantly related plant species such as grasses, trees, or algae. Our initial discovery has opened up the possibility to manipulate levels of natural products in many different areas of plant biotechnology.

[00189] This system is the first ever chloroplast-derived CFPS system capable of transcription and translation and is more high-yielding than previously reported translation-only systems. While other plant-derived systems have been published (wheat germ extract and BY-2 extract), these systems are not suited to prototyping for the chloroplast, which is a more stable transformation cite than the nucleus. This system will enable technological discovery by generating a method for rapid prototyping in a plant context.

[00190] Results

[00191] We set out to design a system for rapid plastid prototyping using a chloroplast extract CFPS system. Figure 29 provides a schematic illustration of plastid prototyping using a chloroplast cell-free system and subsequent chloroplast transformation to prepare modified plants. As illustrated, a library of modified genetic components (e.g., ribosome binding sites (RBS)) comprising > 10³ or 10⁴ members can be screened using a chloroplast CFPS system in order to identify members with desirable characteristics (e.g., optimal protein production). After the components are identified, the components can be utilized to prepare expression vectors for chloroplast transformation.

[00192] We set out to devise a model for designing and testing ribosome binding sites (RBS) based on binding strengths. Figure 30 illustrates a thermodynamic model that provides the basis for designing ribosome binding sites (RBS) that have different strengths. The thermodynamic model is based on the interaction between an mRNA transcript and the 30S ribosomal complex. This thermodynamic model is part of our so- called "RBS calculator." Our RBS calculator can be used as a computation tool that can design RBS's with different binding strengths. The amount of protein expressed in CFPS systems typically correlates with RBS strength. Our RBS calculator can be used for chloroplasts because many plastid 5'-UTR's have Shine Delgamo-like RBS's.

[00193] We created an expression library of 5'-UTRs operably linked to an expressed protein comprising the first 99 nucleotides of one of two biosynthetic enzymes A (Kas) and B (AroG) (from a plant biosynthetic pathway for capsaicin biosynthesis) fused to luciferase. Figure 31 illustrates the results of experiments in which protein expression was measured versus ribosome binding strength for the library of 5'-UTR's. A strong correlation between RBS strength and measured protein output was observed.

[00194] We also assessed protein expression of the full-length enzymes A (Kas) and B (AroG) in our chloroplast extract CFPS system. Figure 32 illustrates the results of experiments in which protein expression was measured versus ribosome binding strength for a library of expression vectors having different ribosome binding sites (RBS) and expressing one of biosynthetic enzymes A or B. Protein production was measure via incorporation of radioactive leucine. Twenty eight (28) variants with different RBS binding strengths were tested in less than four (4) weeks.

[00195] Our methods for assessing genetic components such as RBS also could be automated to increase throughput. Figure 33 illustrates how a chloroplast extract cell-free system can be adapted as part of an automated system. Yields of protein (GFP) in a chloroplast extract using an RBS library and a 16S rRNA sequence from the chloroplast illustrate how a chloroplast cell-free system can be utilized to rank the efficiency of genetic parts such as RBS in an automated system.

[00196] We also assessed whether we could introduce further modifications in our expression template to improve protein production such as codon optimization. Figure 34 illustrates schematically how the effect of codon optimization on protein production can be assessed using a chloroplast cell-free system and how an algorithm can be devised from results in a chloroplast cell-free system to provide an algorithm for codon optimization. Our chloroplast extract CFPS system can then be utilized to assess protein production based on codon optimization prior to transformation of a chloroplast with an expression vector.

[00197] We also assessed how we might utilize our chloroplast extract CFPS system to assess and tune expression of multi-gene clusters. Figure 35 illustrates RT- qPCR analysis of a multi-genic cluster in a chloroplast cell-free system. A cluster (Klebsiella‘ refactored’ v2.) composed of (sixteen) 16 genes (20kb) was used as template for chloroplast cell-free reactions. Reactions were incubated 25°C during lh and 100 pL of the reaction mix was harvested for total RNA extraction. Total RNA was assayed with RT-qPCR. All 16 genes were detected in both cell-free systems, demonstrating that the chloroplast cell-free is able to express multi-gene cluster. We then compared expression in vivo to our results obtained in our CFPS system. Figure 36 provides a comparison of multi-gene expression per Figure 36 versus multi-gene expression in vivo which demonstrates a strong correlation, indicating that our CFPS system can be utilized to predict and tune expression after chloroplast transformation.

[00198] We compared expression of our sixteen (16) multi-gene cluster in a chloroplast extract CFPS system versus an E. coli extract CFPS system. Figure 37 illustrates RT-qPCR analysis of a multi-genic cluster in chloroplast and E. coli cell-free systems. All 16 genes were detected in both cell-free systems, demonstrating that the chloroplast cell-free is able to express a multi-gene cluster. However, the observed correlation between RBS binding and protein production was stronger in our chloroplast extract CFPS system than in a CFPS that utilized an E. coli extract. (See Figure 38).

[00199] We also assessed whether chloroplast in vivo gene expression would correlate with chloroplast cell-free data. (See Figure 39). First, we integrated three (3) engineered nitrogenase (nif) clusters (Klebsiella vl.0, v2.0, and v3.2) into the tobacco plastome under expression from T7 promoters. (See Figure 39 a) and b)). Upon theophylline (Theo) induction, plastome integrated T7 RNAP transcribed the nif clusters. We observed a good correlation between expression in vivo and expression in our chloroplast extract CFPS system.

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[00247] Patent Documents

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US6518058; US6783957; US6869774; US6994986; US7118883; US7189528;

US7312049; US7338789; US7387884; US7399610; US8357529; US8574880;

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[00249] U.S. Patent Publications: US20020058303; US2002034559;

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[00250] Published International Applications: W02003056914A1;

W02004013151A2; W02004035605A2; W02006102652A2; W02006119987A2;

W02007120932A2; WO2014144583; and WO2017117539; the contents of which are incorporated herein by reference in their entirety. [00251] In the foregoing description, it will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention. Thus, it should be understood that although the present invention has been illustrated by specific embodiments and optional features, modification and/or variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[0034] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[00252] Citations to a number of patent and non-patent references are made herein.

The cited references are incorporated by reference herein in their entireties. In the event that there is an inconsistency between a definition of a term in the specification as compared to a definition of the term in a cited reference, the term should be interpreted based on the definition in the specification.

Claims

CLAIMS We claim:

1. A cell-free protein synthesis platform for in vitro transcription of mRNA and/or translation of polypeptides, the platform comprising as a component:

(a) a chloroplast extract prepared from plants, optionally plants that are grown for about 4-8 weeks, optionally wherein the plants are grown in about 6 hours dark and in about 18 hours light during the 4-8 weeks, and optionally wherein the plants are incubated in total dark for 24 hours, preferably 10-12 hours before preparing the chloroplast extract;

and the platform optionally comprising one or more of the following components:

(b) a reaction buffer;

(c) an RNA polymerase; and

(d) the transcription template,

wherein the RNA polymerase is capable of transcribing the transcription template to form a translation template and the chloroplast extract can sustain protein synthesis through a combined transcription/translation reaction.

2. The platform of claim 1, wherein the chloroplast extract is prepared from a species selected from Nicotiana spp..

3. The platform of claim 1, wherein the chloroplast extract is prepared from a species selected from Maize spp., Glycine spp., Oryza spp., and Triticum spp..

4. The platform of claim 1, wherein the chloroplast extract is prepared from a species selected from Solanum spp..

5. The platform of claim 1, wherein the chloroplast extract is prepared from a plant that is engineered to be deficient in a negative effector for in vitro transcription and translation.

6. The platform of claim 1, wherein the chloroplast extract is prepared from a plant that has been engineered to express T7 RNA polymerase in the chloroplast or in the nucleus and T7 RNA polymerase is targeted to the chloroplast.

7. The platform of claim 1, wherein the platform or any component of the platform is preserved, optionally wherein the platform or any component of the platform is preserved through freeze-drying.

8. A method for in vitro transcription of mRNA and/or translation of mRNA to prepare a polypeptide, the method comprising reacting a reaction mixture comprising:

(a) the cell-free protein synthesis platform of claim 1 ;

(b) a template for transcription of the mRNA and/or a template for translation to prepare the polypeptide;

(c) monomers for synthesis of the mRNA and/or the polypeptide;

(d) co-factors, enzymes and/or other reagents necessary for the transcription and/or translation; and

(e) magnesium at a concentration of from about 5 mM to about 20 mM.

9. The method of claim 8, wherein the monomers comprise amino acids at a concentration of between about 0.5 to 4 mM, and preferably at a concentration of about 2 mM.

10. The method of claim 8, wherein the reaction mixture further comprises potassium at a concentration of about 0-200 mM, and preferably at a concentration of about 100 mM.

11. The method of claim 8, wherein the reaction mixture further comprises salts at a total concentration of between about 50-400 mM.

12. The method of claim 8, wherein the reaction mixture further comprises an energy regeneration system that comprises creatine kinase and creatine phosphate.

13. The method of claim 8, wherein the reaction mixture further comprises at least one macromolecular crowding agent.

14. The method of claim 8 wherein the chloroplast extract is prepared by a method that includes a step of adding glycerol to the chloroplast prior to lysing the chloroplasts, and preferably a step of adding glycerol at a concentration of about 5-15% or about 10% to the chloroplasts prior to lysing the chloroplasts.

15. The method of claim 8, wherein the template for the mRNA includes modifications in the 3’UTR that facilitate efficient transcription and/or translation.

16. The method of claim 8, wherein the template for the mRNA includes modifications in the 5’UTR that facilitate efficient transcription and/or translation.

17. The method of claim 8, wherein the reaction mixture further comprises a DNA-dependent RNA polymerase.

18. The method of claim 17, wherein the DNA-dependent RNA polymerase is a bacteriophage DNA-dependent RNA, such as T7 RNA polymerase.

19. The method of claim 8, wherein the method is performed as a batch reaction.

20. The method of claim 8, wherein the method is performed as a semi- continuous reaction.

21. The method of claim 8, wherein the method is performed using automation.

22. The method of claim 8, wherein the method is performed at a temperature between about 20-40 °C.

23. The method of claim 8, wherein the reaction mixture or any component of the reaction mixture is preserved, optionally wherein the reaction mixture or any component of the reaction mixture is preserved through freeze-drying.

24. The method of claim 8, wherein the reaction mixture further comprises an RNAse inhibitor.

25. A method comprising:

(a) creating a test library of genetic parts or components of plants; and

(b) testing the function of the genetic parts or components of the test library in the cell-free protein synthesis platform of claim 1 ; and

(c) optionally assessing gene expression, and optionally using information obtained from assessing gene expression to modify gene expression in a plant; and

(d) optionally wherein the method is used to characterize and assess the genetic parts or components and fine-tune gene expression prior to studying the genetic parts and gene expression in plants.

26. A method comprising:

(a) creating a test library of codon-optimized constructs of a gene product; and

(b) testing expression of the codon-optimized constructs of the test library in the cell-free protein synthesis platform of claim 1 ; and (c) optionally using information obtained from testing expression of the codon-optimized constructs to modify expression in a plant; and

(d) optionally wherein the method is used to characterize and assess codon optimization and fine-tune of gene expression prior to studying codon optimization and fine-tuning of gene expression in plants.

27. A method comprising:

(a) creating a test library of constructs expressing several genes; and

(b) testing expression of the constructs of the test library in the cell-free protein synthesis platform of claim 1; and

(c) optionally using information obtained from testing expression of the construct to modify gene expression in a plant; and

(d) optionally wherein the method is used to is used to provide information to a geneticist regarding the design of multi gene functions.

28. A method comprising:

(a) creating a test library of constructs of a biosynthetic pathway expressing enzymes; and

(b) testing the constructs of the test library in the cell-free protein synthesis platform of claim 1 ; and.

(c) assessing gene expression, metabolite production, requirement of accessory proteins or other molecules, and/or metabolic parameters;

(d) optionally wherein the method is used to aid design of multi gene functions; and (e) optionally wherein the method is used to aid the design and function of enzyme pathways for example in order to fine- tune expression levels of the enzymes and testing possible accessory proteins or other molecules required for proper pathway function.

29. A method comprising:

(a) creating plant specific sensors comprising genetic circuits that respond to external commands; and

(b) testing the sensors in the cell-free protein synthesis platform of claim 1 ; and.

(c) assessing sensor response and circuit behavior; and

(d) optionally wherein the method is used to aid the design and function of sensor and genetic circuits.

30. A kit comprising as components: (a) a cellular extract prepared from a plant chloroplast, optionally wherein the cellular extract is preserved, such as for example by freeze-drying; and (b) a reaction mixture for transcribing an mRNA from a DNA template and or for translating an mRNA to prepare a polypeptide, optionally wherein the reaction mixture is preserved, such as for example by freeze-drying.

31. The kit of claim 30, wherein the plant is selected from Nicotiana tabacum, Maize spp., Glycine spp., Oryza spp., Triticum spp. and Solanum spp..

32. The kit of claim 30, wherein the reaction mixture comprises one or more components selected from the group consisting of amino acids which optionally may include non-canonical amino acids, NTPs, salts, cofactors, an energy source and optionally an energy source comprising a phosphate group.

33. A method for preparing an extract from chloroplasts of a plant, the method comprising: (a) obtaining a plant, optionally a plant that has been grown for about 4-8 weeks and preferably about 6 weeks;

(b) isolating the chloroplasts from the plant;

(c) adding a polyol compound to the isolated chloroplasts, optionally at a concentration of about 5-15% and preferably at a concentration of about 10%; and

(d) lysing the chloroplasts, optionally by freezing, and

separating the extract from the lysed chloroplasts, optionally separating a soluble extract from the lysed chloroplasts.