Single-molecule in vitro evolution
First Claim
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1. A method for isolating one or more genetic elements encoding a gene product having a desired activity, comprising the steps of:
- a) providing a population of genetic elements and expressing the genetic elements to produce their respective gene product(s), such that each molecule of gene product is linked to the genetic element encoding it at a ratio of one molecule of gene product per genetic element or less;
b) compartmentalising the genetic elements into microcapsules; and
c) sorting the genetic elements according to the activity of the gene product.
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Abstract
The invention relates to a method for isolating one or more genetic elements encoding a gene product having a desired activity, comprising the steps of: a) providing a population of genetic elements and expressing the genetic elements to produce their respective gene product(s), such that each molecule of gene product is linked to the genetic element encoding it at a ratio of one molecule of gene product per genetic element or less; b) compartmentalising the genetic elements into microcapsules; and c) sorting the genetic elements according to the activity of the gene product.
91 Citations
28 Claims
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1. A method for isolating one or more genetic elements encoding a gene product having a desired activity, comprising the steps of:
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a) providing a population of genetic elements and expressing the genetic elements to produce their respective gene product(s), such that each molecule of gene product is linked to the genetic element encoding it at a ratio of one molecule of gene product per genetic element or less;
b) compartmentalising the genetic elements into microcapsules; and
c) sorting the genetic elements according to the activity of the gene product. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22)
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23. A phosphotriesterase having a kcat of 105 s−
- 1 or more.
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24. A phosphotriesterase having a kcat of 2.8×
- 105 S−
1.
- 105 S−
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25. A phosphotriesterase comprising one or more of the mutations selected from the group consisting of:
- I106T and F132L;
I106S, F132L, S308L and Y309R;
I106S;
I106L; and
I106D, W131Y and F132S.
- I106T and F132L;
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26. A method for preparing a gene product, comprising the steps of:
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(a) preparing a genetic element encoding the gene product;
(b) compartmentalising genetic elements into microcapsules;
(c) expressing the genetic elements to produce their respective gene products within the microcapsules and linking the gene products to the genetic elements, such that each genetic element is linked to not more than one molecule of its respective gene product;
(d) sorting the genetic elements which produce the gene product(s) having the desired activity; and
(e) expressing the gene product having the desired activity.
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27. A method for screening a compound or compounds capable of modulating the activity of a gene product, comprising the steps of:
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(a) preparing a repertoire of genetic elements encoding gene product;
(b) compartmentalising the genetic elements into microcapsules;
(c) expressing the genetic elements to produce their respective gene products within the microcapsules and linking the gene products to the genetic elements, such that each genetic element is linked to not more than one molecule of its respective gene product;
(d) sorting the genetic elements which produce the gene product(s) having the desired activity; and
(e) contacting a gene product having the desired activity with the compound or compounds and monitoring the modulation of an activity of the gene product by the compound or compounds.
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28. A method for preparing a compound or compounds comprising the steps of:
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(a) providing a synthesis protocol wherein at least one step is facilitated by a polypeptide;
(b) preparing genetic elements encoding variants of the polypeptide which facilitates this step;
(c) compartmentalising the genetic elements into microcapsules;
(d) expressing the genetic elements to produce their respective gene products within the microcapsules and linking the gene products to the genetic elements, such that each genetic element is linked to not more than one molecule of its respective gene product;
(e) sorting the genetic elements which produce polypeptide gene product(s) having the desired activity; and
(f) preparing the compound or compounds using the polypeptide gene product identified in (e) to facilitate the relevant step of the synthesis.
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Specification