Optical sorting method

Abstract

The invention describes a method for isolating one or more genetic elements encoding a gene product having a desired activity, comprising the steps of: (a) compartmentalising genetic elements into microcapsules; (b) expressing the genetic elements to produce their respective gene products within the microcapsules; (c) sorting the genetic elements which produce the gene product having the desired activity using a change in the optical properties of the genetic elements. The invention enables the in vitro evolution of nucleic acids and proteins by repeated mutagenesis and iterative applications of the method of the invention.

Description

[0001] This application is a Continuation-in-Part of International Application No. PCT / GB00 / 00030, filed Jan. 6, 2000, designating the United States, and claims the priority of United Kingdom Application No.: GB 9900298.2, filed Jan. 7, 1999.[0002] The present invention relates to methods for use in in vitro evolution of molecular libraries. In particular, the present invention relates to methods of selecting nucleic acids encoding gene products in which the nucleic acid and the activity of the encoded gene product are linked by compartmentation. [0003] Evolution requires the generation of genetic diversity (diversity in nucleic acid) followed by the selection of those nucleic acids which result in beneficial characteristics. Because the nucleic acid and the activity of the encoded gene product of an organism are physically linked (the nucleic acids being confined within the cells which they encode) multiple rounds of mutation and selection can result in the progressive survival of ...

AI Technical Summary

Benefits of technology

[0026] The genetic element and the gene product thereby encoded are linked by confining each genetic element and the respective gene product encoded by the genetic element within the same microcapsule. In this way the gene product in one microcapsule cannot cause a change in any other microcapsules. In addition, further linking means may be employed to link gene products to the genetic elements encoding them, as set forth below.

[0028] Preferably, the microcapsules used in the method of the present invention will be capable of being produced in very large numbers, and thereby to compartmentalise a library of genetic elements which encodes a repertoire of gene products.

[0037] Additionally, the genetic elements isolated after a first round of sorting may be subjected to mutagenesis before repeating the sorting by iterative repetition of the steps of the method of the invention as set out above. After each round of mutagenesis, some genetic elements will have been modified in such a way that the activity of the gene products is enhanced (i.e., measurable activity increased by at least 10% relative to wild-type).

Problems solved by technology

Thus, the practical limitation on library size allowed by phage display technology is of the order of 107 to 1011, even taking advantage of λ phage vectors with excisable filamentous phage replicons.

However, the scope of the above systems is limited to the selection of proteins and furthermore does not allow direct selection for activities other than binding, for example catalytic or regulatory activity.

However, selection for “catalytic” or binding activity using SELEX is only possible because the same molecule performs the dual role of carrying the genetic information and being the catalyst or binding molecule (aptamer).

Additionally, proteins may not be selected using the SELEX procedure.

The range of catalysts, substrates and reactions which can be selected is therefore severely limited.

However, none of the methods so far developed have provided molecules of comparable diversity and functional efficacy to those that are found naturally.

Additionally, there are no man-made “evolution” systems which can evolve both nucleic acids and proteins to effect the full range of biochemical and biological activities (for example, binding, catalytic and regulatory activities) and that can combine several processes leading to a desired product or activity.

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