Methods and compositions for cell-free biological reactions
Compositions and methods disclosed herein relate to improved in vitro cell-free systems for various biological reactions. In one aspect, a composition of the present disclosure includes: a cell-free extract derived from an organism; a nucleic acid; and an organelle that is heterologous to the organism.
1 . A composition, comprising:
a cell-free extract obtained from an organism, wherein said organism is E. coli;
a nucleic acid; and
an organelle that is heterologous to the organism, wherein said heterologous organelle is a mitochondria.
2 . The composition of claim 1 , wherein the cell-free extract is present at a concentration of about 0.005-1000 mg/ml, 0.05-500 mg/ml, 0.5-100 mg/ml, or 1-20 mg/ml, wherein the extract has been at least partially purified.
3 . The composition of claim 1 , wherein the nucleic acid comprises a linear DNA or a plasmid DNA or an RNA.
4 . The composition of claim 1 , wherein the nucleic acid comprises a template nucleic acid for expression into a desired RNA or protein product.
5 . The composition of claim 1 , wherein the organelle comprises intact organelle and/or non-intact organelle, wherein in the non-intact organelle, an outer membrane and/or inner membrane of the organelle have been at least partially disrupted.
6 . The composition of claim 1 , wherein the organelle is present at a concentration of about 0.000015-100 mg/ml, 0.00015-50 mg/ml, 0.0015-10 mg/ml, or 0.15-4.5 mg/ml.
7 . The composition of claim 1 , wherein the organelle is at least partially purified, and is stored in a solution free of inhibitors to cell-free transcription and translation.
8 . The composition of claim 1 , wherein a ratio of the extract to the organelle by weight is between about 100,000,000:1 and 0.00005:1; between 600,000:1 and 0.005:1; between 300:1 and 0.05:1; or between 10:1 and 0.2:1.
9 . The composition of claim 1 , wherein the organelle is obtained from eukaryotic cells.
10 . The composition of claim 1 , further comprising cofactors, enzymes, and other reagents for transcription and/or translation.
11 . The composition of claim 1 , further comprising a substrate capable of entering the TCA cycle.
12 . The composition of claim 1 , wherein said mitochondria is from Nicotiana tabacum cv. BY-2 cells.