System for continuous mutagenesis in vivo to facilitate directed evolution

Patent metadata

Field	Value
Publication number	US-10760071-B2
Application number	US-201615747282-A
Country	US
Kind code	B2
Filing date	Jul 25, 2016
Priority date	Jul 24, 2015
Publication date	Sep 1, 2020
Grant date	Sep 1, 2020

Abstract

A system for continuous mutagenesis to facilitate directed evolution, the system including DNA polymerases carrying the novel K54E point mutation, and other point mutations including I709N, A759R, D424A (herein called K54E_LF Pol I) and this methods of use to produce and detect lines where mutagenesis is continuous and does not exhibit the usual decline in mutagenesis with sequential cloning.

First claim

The invention claimed is:

1. A mutant DNA polymerase with at least 90% homology to SEP ID NO: 2, said polymerase comprising a mutation at position K54, and further comprising mutations at positions I709, A759, and D424, provided that the polymerase displays continuous mutagenesis equivalent to that of a polymerase of SEQ ID NO: 15, wherein the mutation at position I709 is an I709N mutation, wherein the mutation at position A759 is an A759R mutation, and wherein the mutation at position D424 is a D424A mutation.

2. The mutant DNA polymerase of claim 1 , comprising SEQ ID NO: 15.

3. The mutant DNA polymerase of claim 1 , consisting of SEQ ID NO: 15.

4. A polynucleotide that encodes the mutant DNA polymerase of claim 1 .

5. A plasmid comprising the polynucleotide of claim 4 operably linked to a first promoter.

6. An E. coli cell comprising the plasmid of claim 5 .

7. A method of performing directed evolution of a gene of interest, the method comprising:
transforming the E. coli cell of claim 6 with a reporter plasmid, wherein the reporter plasmid comprises a polynucleotide that encodes an unmutated gene of interest, thereby performing continuous mutagenesis on the gene of interest and producing an E. coli cell comprising a reporter plasmid that includes a mutant form of the gene of interest,
purifying the reporter plasmid that includes the mutant form of the gene of interest from the E. coli cell, thereby creating a purified mutated reporter plasmid;
transforming an E. coli cell with the purified mutated reporter plasmid; and
determining the activity of the mutant form of the gene of interest relative to the unmutated gene of interest.

8. The method of claim 7 , wherein the gene of interest comprises a fluorescent protein.

9. The method of claim 8 , wherein the gene of interest comprises GFP.

Assignees

• Univ California

Inventors

• Camps Manel

Classifications

• C12N9/1252 Primary

  DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase · CPC title

• C12N15/102

  Mutagenizing nucleic acids · CPC title

• C12N15/1024 Primary

  In vivo mutagenesis using high mutation rate "mutator" host strains by inserting genetic material, e.g. encoding an error prone polymerase, disrupting a gene for mismatch repair · CPC title

• C12P19/34

  Polynucleotides, e.g. nucleic acids, oligoribonucleotides · CPC title

• C12N15/70

  Vectors or expression systems specially adapted for E. coli · CPC title