Fluorescence-based reporters for mutagenesis detection in E. coli

Patent metadata

Field	Value
Publication number	US-11788113-B2
Application number	US-202217728812-A
Country	US
Kind code	B2
Filing date	Apr 25, 2022
Priority date	Jan 1, 2017
Publication date	Oct 17, 2023
Grant date	Oct 17, 2023

Abstract

Direct detection of mutagenesis in prokaryotes by reversion of an inactivating mutation (reversion mutation assay), producing a quantitative signal for in vivo mutagenesis, may greatly reduce the amount of test chemicals and labor involved in these assays. Further, transcriptional coupling of β-lactamase reversion and GFP, translational fusion between β-lactamase and GFP with stop codon in GFP, and a novel dual reporter to monitor continuous mutagenesis may be used in methods described herein.

First claim

What is claimed is:

1. A method of detecting mutagenesis in Escherichia coli ( E. coli ), the method comprising:
(a) culturing E. coli cells in a first liquid culture at a restrictive temperature, wherein the E. coli cells in the first liquid culture comprise a plasmid, wherein the plasmid comprises (i) a first polynucleotide encoding a non-fluorescent protein comprising the amino acid sequence of SEQ ID NO:11, and (ii) a second polynucleotide encoding an active β-lactamase, wherein the first polynucleotide and the second polynucleotide are operably linked to a first promoter,
wherein the E. coli cells in the first liquid culture further comprise
(i) an error prone polymerase I operably linked to a second promoter, where the second promoter promotes transcription of the error prone polymerase I at the restrictive temperature, and
(ii) a wild type polymerase I that is operably linked to a third promoter, wherein the third promoter promotes transcription of the error prone polymerase I at a permissive temperature;

(b) plating the E. coli cells in the first liquid culture on a solid media comprising a β-lactam antibiotic;
(c) incubating the solid media at the permissive temperature to allow the growth of E. coli colonies;
(d) selecting a fluorescent E. coli colony from the solid media;
(e) culturing the fluorescent E. coli colony in a second liquid culture at the permissive temperature, wherein the second liquid culture comprises the β-lactam antibiotic; and
(f) measuring a change in fluorescence of the second liquid culture relative to the first liquid culture,
wherein the change in fluorescence indicates mutagenesis of the non-fluorescent protein to a fluorescent protein.

2. The method of claim 1 , wherein the first polynucleotide is located 5′ to the second polynucleotide in the plasmid.

3. The method of claim 1 , wherein the plasmid further comprises a linker between the first polynucleotide and the second polynucleotide.

4. The method of claim 1 , wherein the β-lactam antibiotic is carbenicillin.

5. The method of claim 1 , wherein the β-lactamase is TEM-1.

6. The method of claim 1 , wherein the fluorescent protein comprises GFP.

7. The method of claim 1 , wherein the fluorescent protein comprises the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10.

8. The method of claim 1 , wherein the first polynucleotide and the second polynucleotide are expressed as a fusion protein.

9. The method of claim 1 , further comprising exposing the E. coli cells to a test compound added to the first liquid culture.

10. The method of claim 9 , wherein the test compound is a mutagen.

Assignees

• Univ California

Inventors

• Camps Manel
• Allen Jennifer

Classifications

• C12Q1/34 Primary

  involving hydrolase · CPC title

• C12N9/86

  acting on amide bonds in cyclic amides, e.g. penicillinase {(3.5.2)} · CPC title

• C12N15/72

  Expression systems using regulatory sequences derived from the lac-operon · CPC title

• C12Y305/02006

  Beta-lactamase (3.5.2.6) · CPC title

• C12N15/70 Primary

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