Patent ID: 7888293
Filing Date: 2011-02-15
Classification: C12N

Abstract:
1. A method of selecting an orthogonal mRNA orthogonal rRNA pair, the method comprising: a) providing a first library encoding mRNA molecules, the individual mRNA members encoded by which comprise a sequence coding for a fusion polypeptide, said fusion polypeptide comprising a positive selectable marker polypeptide and a negative selectable marker polypeptide, wherein for individual members of said first library, said sequence coding for a fusion polypeptide is operably linked to one of a plurality of mutated ribosome binding sites; b) providing a second library encoding mutant rRNA molecules, the individual members of which are mutated in a region comprising sequence that interacts with mRNA at the ribosome binding site; c) introducing said first library to cells and performing a negative selection against mRNA molecules encoded by said first library that are substrates for wild-type ribosomes, thereby selecting members of said first library that encode mRNAs that are not substrates for wild-type ribosomes; d) introducing said second library encoding mutant rRNA molecules into cells comprising said first library members selected in step (c), and performing a positive selection for those cells expressing said positive selectable marker, whereby an orthogonal mRNA orthogonal rRNA pair comprising a first library member encoding an mRNA that is efficiently translated by a ribosome comprising an rRNA mutant encoded by second library is identified.