Vector-based mutagenesis system

Abstract

Strategies, reagents, methods, and systems for modulating the mutation rate in cells are provided herein. The strategies, reagents, methods, and systems are broadly applicable for the modulation of mutation rates in cells where high mutation rates and/or control over a broad range of mutation rates is desired, for example, in the context of diversifying a nucleic acid sequence or a plurality of such sequences within a population of cells, for the generation of diversified nucleic acid libraries, and for directed evolution of nucleic acids and encoded products.

1 - 125 . (canceled) 
     
     
         126 . An expression construct for modulating the mutation rate of nucleic acids in a bacterial cell, the construct comprising:
 (i) a drift promoter; and   (ii) a nucleic acid sequence encoding one or more gene products selected from a gene product that disrupts a proofreading pathway, a translesion synthesis pathway, a methyl-directed mismatch repair pathway, a base excision repair pathway, or a base selection pathway of the bacterial cell, or any combination thereof,   wherein the nucleic acid sequence encoding the one or more gene products is under the control of the drift promoter.   
     
     
         127 . The expression construct of  claim 126 , wherein the gene product that disrupts a proofreading pathway is a dnaQ926, BRM1, BR11, BR1, BR6, or BR13 gene product. 
     
     
         128 . The expression construct of  claim 126 , wherein the gene product that disrupts a translesion synthesis pathway is an umuD′, umuC, recA, dinB, or polB gene product. 
     
     
         129 . The expression construct of  claim 126 , wherein the gene product that disrupts a methyl-directed mismatch repair pathway is a mutS, mutL, mutH, dam, or seqA gene product. 
     
     
         130 . The expression construct of  claim 126 , wherein the gene product that disrupts a base excision repair pathway is a ugi, AID, APOBEC, CDA, MAG, or AAG gene product. 
     
     
         131 . The expression construct of  claim 126 , wherein the gene product that disrupts a base selection pathway is a dnaE74, dnaE486, dnaE1026, dnaX36, dnaX2016, emrR, nrdAB, nrdA(H59A)B, nrdA(A65V)B, nrdA(A301V)B, nrdAB(P334L), or nrdEF gene product. 
     
     
         132 . The expression construct of  claim 126 , wherein the expression construct further comprises a nucleic acid sequence encoding a rsmE, cchA, yffI, or yfjY gene product. 
     
     
         133 . The expression construct of  claim 126 , wherein the expression construct comprises a nucleic acid sequence encoding a dnaQ926 gene product. 
     
     
         134 . The expression construct of  claim 126 , wherein the expression construct comprises a nucleic acid sequence encoding a dnaE74, a dnaE486, a dnaE1026, a dnaX36, a dnaX2016, a rpsD12, a rpsD14, a rpsD16, a polB, a polB(D156A), a MAG1, a AAG(Y127I-H136L), and/or a Δ80-AAG(Y127I-H136L) gene product. 
     
     
         135 . The expression construct of  claim 126 , wherein the one or more gene product is dnaQ926, umuD′, umuC, recA730, dam, seqA, emrR, PBS2, UGI, or CDA1, or any combination thereof. 
     
     
         136 . The expression construct of  claim 126 , wherein the expression construct comprises a nucleic acid sequence encoding a: dnaQ926 gene product, a dam gene product, a seqA gene product, an emrR gene product, a ugi gene product, and a CDA1 gene product. 
     
     
         137 . The expression construct of  claim 126  comprising the sequence set forth in any one of SEQ ID NO: 27, 28, 29, 33, 34, and 35. 
     
     
         138 . The expression construct of  claim 126 , wherein the drift promoter comprises the sequence set forth in SEQ ID NO: 124. 
     
     
         139 . The expression construct of  claim 126 , wherein the expression construct comprises a nucleic acid sequence encoding an arabinose operon regulatory protein. 
     
     
         140 . The expression construct of  claim 139 , wherein the nucleic acid sequence encoding an arabinose operon regulatory protein comprises the sequence set forth in SEQ ID NO: 36. 
     
     
         141 . A cell comprising the expression construct of  claim 126 . 
     
     
         142 . A plasmid comprising the expression construct of  claim 126 . 
     
     
         143 . The plasmid of  claim 142 , wherein the plasmid comprises a bacterial origin of replication. 
     
     
         144 . The plasmid of  claim 143 , wherein the origin of replication is a cloDF13 origin of replication. 
     
     
         145 . The plasmid of  claim 142  further comprising an expression construct comprising a nucleic acid sequence encoding pIII protein.