Methods and Compositions for Rapid Assembly of Genetic Modules

Abstract

Provided herein are methods and compositions for rapid assembly of genetic modules, as well as seamless transition from in vitro to in vivo testing of genetic constructs.

1 . A non-naturally occurring library of genetic modules, comprising:
 a plurality of pre-designed promoters,   a plurality of pre-designed untranslated regions,   a plurality of pre-designed terminators,   a plurality of pre-designed stage 1 vectors, and   at least one pre-designed stage 2 vector,   wherein each promoter, untranslated region, terminator, stage 1 vector and stage 2 vector are engineered to have a pair of restriction sites of a first type IIs enzyme;   wherein the promoters, untranslated regions and terminators are designed to be assembled into a plurality of recombinant transcription units Tu1, Tu2 and TuN wherein N>=3, each recombinant transcription unit being present in a separate stage 1 vector and flanked by a first pair of restriction sites of the first type IIs enzyme, wherein the first pair of restriction sites for each recombinant transcription unit are pre-designed such that upon digestion by the first type IIs enzyme, compatible cohesive ends are generated to allow ligation of the recombinant transcription units into a linear DNA molecule having a predetermined order 5′-Tu1-TuN-Tu2-3′;   wherein each stage 2 vector has a second pair of restriction sites of the first type IIs enzyme, wherein the second pair of restriction sites are pre-designed such that upon digestion by the first type IIs enzyme, a first and second cohesive end are generated to allow direct ligation of the first cohesive end with Tu1 at its 5′ end and direct ligation of the second cohesive end with Tu2 at its 3′ end.   
     
     
         2 . The library of  claim 1 , wherein N=<9. 
     
     
         3 . The library of  claim 1 , wherein the first type IIs enzyme is selected from BsaI, Eco31I, BspTN1, Bso31I, BbsI, BpuAI, BpiI, BstV21, BsmBI, Esp3I, FokI, AlwI, and BfilI. 
     
     
         4 . The library of  claim 1 , wherein each recombinant transcription unit comprises a promoter, an untranslated region, a coding sequence and a terminator. 
     
     
         5 . The library of  claim 1 , wherein each promoter, untranslated region, terminator, stage 1 vector and stage 2 vector further comprise flanking restriction sites of a second type IIs enzyme. 
     
     
         6 . The library of  claim 5 , wherein the second type IIs enzyme is selected from BsaI, Eco31I, BspTN1, Bso31I, BbsI, BpuAI, BpiI, BstV21, BsmBI, Esp3I, FokI, AlwI, and BfilI. 
     
     
         7 . The library of  claim 1 , wherein each stage 1 vector comprises a different origin of replication and/or a different selectable marker. 
     
     
         8 . The library of  claim 7 , wherein the origin of replication is selected from colE1, pSC101, p15A, pBBR1, pMB1 and R6K. 
     
     
         9 . The library of  claim 7 , wherein the selectable marker is selected from AmpR, KanR, CmR, ZeoR, TetR, SpecR, StrepR, NeoR, and BleR. 
     
     
         10 . The library of  claim 1 , wherein the stage 2 vector further comprises a pair of restriction sites of a second type IIs enzyme that flank the second pair of restriction sites of the first type IIs enzyme. 
     
     
         11 . The library of  claim 1 , wherein each stage 2 vector comprises a different origin of replication and/or a different selectable marker. 
     
     
         12 . The library of  claim 1 , further comprising a first primer and second primer that are designed to span the first and second cohesive end, respectively. 
     
     
         13 . The library of  claim 12 , wherein the first primer partially anneals with the stage 2 vector at Tm<40° C. and partially with Tu1 at Tm<40° C., and the second primer partially anneals with the stage 2 vector at Tm<40° C. and partially with Tu2 at Tm<40° C. 
     
     
         14 . A kit for in vitro assembly of genetic modules, comprising:
 (a) the library of  claim 1 , and   (b) instruction for in vitro assembly of a coding sequence of interest with a promoter, an untranslated region and a terminator selected from the library into a transcription unit in a stage 1 vector, and further assembly of a plurality of transcription units into a stage 2 vector.