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.

1 . A method for isolating one or more genetic elements encoding a gene product having a desired activity the expression of which may result, directly or indirectly, in the modification of an optical property of a genetic element encoding the gene product, comprising the steps of: 
 (a) expressing the genetic elements to produce their respective gene products such that the gene products are linked to the genes encoding them;    (b) compartmentalising genetic elements into microcapsules; and    (c) sorting the genetic elements which produce the gene product(s) having the desired activity according to a change in the optical properties of the genetic elements.    
     
     
         2 . The method according to  claim 1 , wherein the activity of the desired gene product results, directly or indirectly, in the modification of the genetic element encoding the gene product to enable the isolation of the genetic element.  
     
     
         3 . The method according to  claim 2 , wherein in the modification of the genetic element induces a change in its optical properties.  
     
     
         4 . The method according to  claim 2 , wherein the modification of the genetic element enables it to be further modified outside the microcapsule so as to induce a change in its optical properties.  
     
     
         5 . The method according to  claim 2 , wherein a part of the genetic element is a ligand and the desired gene product binds, directly or indirectly, to said ligand to enable the isolation of the genetic element.  
     
     
         6 . The method according to  claim 5 , wherein the ligand is also encoded by the genetic element.  
     
     
         7 . The method according to  claim 2 , wherein a part of the genetic element is a substrate and the activity of the desired gene product results, directly or indirectly, in the conversion of said substrate into a product which remains part of the genetic element and enables its isolation.  
     
     
         8 . The method according to  claim 2 , wherein the product of the activity of the desired gene product results, directly or indirectly, in the generation of a product which is subsequently complexed with the genetic element and enables its isolation.  
     
     
         9 . The method according to  claim 1  wherein the activity of the desired gene product results, directly or indirectly, in the alteration of the expression of a second gene within the compartment and the activity of the product of the said second gene enables the isolation of the genetic element using a change in the optical properties of the genetic element.  
     
     
         10 . The method according to  claim 1 , wherein step (a) comprises: 
 expressing the genetic elements to produce their respective gene products, linking the gene products to the genetic elements encoding them and isolating the complexes thereby formed.    
     
     
         11 . The method according to  claim 1 , wherein in step (c) the complexes are directly sorted based on their changed optical properties to isolate genetic elements encoding a gene product having the desired activity.  
     
     
         12 . The method according to  claim 1 , wherein in step (c) the complexes are further reacted to induce a conditional change in optical properties of the genetic element dependent on the presence of gene products with the desired activity in the complex.  
     
     
         13 . The method according to  claim 1 , wherein the complexes are subjected to a further compartmentalisation step in order to isolate the genetic elements encoding a gene product having the desired activity.  
     
     
         14 . The method according to  claim 1 , wherein the change in optical properties of the genetic element is due to binding of a gene product with distinctive optical properties to the genetic element.  
     
     
         15 . The method according to  claim 1 , wherein the change in optical properties of the genetic element is due to binding of a ligand with distinctive optical properties by the gene product.  
     
     
         16 . The method according to  claim 1 , wherein the change in optical properties of the genetic element is due to a change in the optical properties of the gene product when bound to ligand.  
     
     
         17 . The method according to  claim 1 , wherein the change in optical properties of the genetic element is due to a change in the optical properties of the ligand when bound by the gene product.  
     
     
         18 . A method according to  claim 1 , wherein the change in optical properties of the genetic element is due to a change in the optical properties of both ligand and gene product on binding.  
     
     
         19 . The method according to  claim 1 , wherein the change in optical properties of the genetic element is due to the different optical properties of the substrate and the product of the reaction being selected.  
     
     
         20 . The method according to  claim 1 , wherein both substrate and product have similar optical properties, but only the product, and not the substrate of the reaction being selected binds to, or reacts with, the genetic clement, thereby changing the optical properties of the genetic element.  
     
     
         21 . The method according to  claim 1 , wherein further reagents specifically bind to, or specifically react with, the product (and not the substrate) attached to the genetic element, thereby altering the optical properties of the genetic element.  
     
     
         22 . The method according to  claim 1 , wherein a non-desired activity of a gene product results in a change in the optical properties of the genetic element which is distinct from that resulting from the desired activity.  
     
     
         23 . The method according to  claim 22 , wherein the optical change resulting from the non-desired activity is used to negatively select the genetic elements.  
     
     
         24 . The method according to  claim 22 , wherein negative selection is combined with positive selection to improve reaction specificity.  
     
     
         25 . The method according to  claim 24 , wherein the improved reaction specificity is an improvement in binding specificity.  
     
     
         26 . The method according to  claim 24 , wherein the improved reaction specificity is an improvement in regio- and/or stereo-selectivity for substrate and/or product.  
     
     
         27 . The method according to  claim 1 , wherein the genetic elements are isolated from a library of genetic elements encoding a repertoire of gene products.  
     
     
         28 . The method according to  claim 1 , wherein each genetic element encodes two or more genes and each gene product must have a desired activity in order for the optical properties of the genetic element to be modified to enable them to be sorted.  
     
     
         29 . The method according to  claim 1 , wherein each genetic element encodes two or more genes and the gene products must bind to each other in order for the optical properties of the genetic element to be modified and the genetic elements sorted.  
     
     
         30 . The method according to  claim 1  further comprising the additional step of: 
 (d) introducing one or more mutations into the genetic element(s) isolated in step (c).    
     
     
         31 . The method according to  claim 30  further comprising iteratively repeating one or more of steps (a) to (d).  
     
     
         32 . The method according to  claim 1  further comprising amplifying the genetic elements.  
     
     
         33 . The method according to  claim 1 , wherein microencapsulation is achieved by forming a water-in-oil emulsion of the aqueous solution in an oil-based medium.  
     
     
         34 . The method according to  claim 1 , wherein the genetic element comprises the gene attached to a microbead.  
     
     
         35 . The method according to  claim 34 , wherein the microbead is nonmagnetic, magnetic or paramagnetic.  
     
     
         36 . The method according to  claim 1 , wherein the genetic elements are sorted by detection of a change in their fluorescence.  
     
     
         37 . The method according to  claim 36 , wherein the sorting of genetic elements is performed using a fluorescence activated cell sorter (FACS).  
     
     
         38 . The method according to  claim 36 , wherein the different fluorescence properties of the substrate and the product are due to fluorescence resonance energy transfer (FRET).  
     
     
         39 . The method according to  claim 1 , wherein the internal environment of the microcapsules is modified by the addition of one or more reagents to the oil phase.  
     
     
         40 . The method according to  claim 1 , wherein genetic elements modified directly or indirectly by the activity of the desired gene product are further modified by Tyramide Signal Amplification (TSA™; NEN), resulting directly or indirectly in a change in the optical properties of said genetic elements thereby enabling their separation.  
     
     
         41 . A product isolated according to the method of  claim 1 .  
     
     
         42 . A method for preparing a gene product, comprising the steps of: 
 (a) preparing a genetic element encoding the gene product;    (b) expressing the genetic elements to produce their respective gene products;    (c) compartmentalising genetic elements into microcapsules,    (d) sorting the genetic elements which produce the gene product(s) having the desired activity using a change in their optical properties; and    (e) expressing the gene product having the desired activity.    
     
     
         43 . A method for screening a compound or compounds capable of modulating the activity of a gene product, comprising the steps of: 
 (a) preparing a genetic element encoding the gene product;    (b) expressing the genetic elements to produce their respective gene products;    (c) compartmentalising genetic elements into microcapsules;    (d) sorting the genetic elements which produce the gene product(s) having the desired activity using a change in their optical properties; 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.    
     
     
         44 . A method for preparing a compound or compounds comprising the steps of: 
 (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) expressing the genetic elements to produce their respective gene products;    (d) compartmentalising the genetic elements into microcapsules;    (e) sorting the genetic elements which produce polypeptide gene product(s) having the desired activity using a change in their optical properties; and    (f) preparing the compound or compounds using the polypeptide gene product identified in (e) to facilitate the relevant step of the synthesis.