Fluorescence-based reporters for mutagenesis detection in <i>E. coli</i>
11339420 · 2022-05-24
Assignee
Inventors
Cpc classification
C12N15/70
CHEMISTRY; METALLURGY
C12N9/86
CHEMISTRY; METALLURGY
C12N15/72
CHEMISTRY; METALLURGY
International classification
C12N9/86
CHEMISTRY; METALLURGY
C12N15/70
CHEMISTRY; METALLURGY
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.
Claims
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 comprising (i) a first polynucleotide encoding an inactive β-lactamase and having a nucleotide sequence selected from the group consisting of SEQ ID NOS: 2-7, and (ii) a second polynucleotide encoding a fluorescent protein, wherein the first polynucleotide and the second polynucleotide are operably linked to a first promoter, wherein when the first and second polynucleotides are expressed as a fusion and when the fluorescent protein is green fluorescent protein (GFP), the GFP is superfolder GFP and the inactive β-lactamase is located C-terminally to the superfolder GFP in the fusion protein; wherein the E. coli cells 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 first solid media at the permissive temperature; (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 growth of the E. coli cells of the second liquid culture relative to the first liquid culture, wherein the change in growth indicates mutagenesis of the inactive β-lactamase to an active β-lactamase.
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.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1)
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(6)
DETAILED DESCRIPTION OF THE INVENTION
(7) The present disclosure encompasses reporter constructs used for the direct detection of mutagenesis in prokaryotes. In some embodiments, the reporter constructs use transcriptional coupling of β-lactamase (TEM-1) reversion and a fluorescent protein (e.g., GFP). The reporter constructs may also employ translational fusion between β-lactamase (TEM-1) and a fluorescent protein (e.g., GFP) containing a stop codon. In other embodiments, the reporter constructs may be designed to monitor continuous mutagenesis and measure the mutagenesis rate in a mutator strain. The reporters described herein produce a quantitative output, and therefore can reduce the amount of test compounds and labor involved in the performance of mutagenesis assays.
(8) I. Mutagenesis Assays
(9) Mutagenesis may be detected directly or indirectly. Direct detection of mutagenesis can be performed in prokaryotes. Genotoxicity can also be detected indirectly, through transcriptional fusion of a reporter gene to a promoter that is indicative of DNA damage, such as genes belonging to the SOS response (umuDC, sulA, recN, recA), alkA, or nrdA. Genotoxicity can also be detected physically by detecting DNA damage (breaks or rearrangements) using, e.g., Comet assay. In addition to prokaryotic systems, a variety of eukaryotic organisms, notably yeast, Drosophila, and mouse, have been used.
(10) Relative to indirect methods of mutagenesis detection in prokaryotes, mutagenesis assays have the advantage of being able to detect specific changes in DNA sequence rather than all DNA damage-induced alterations in gene expression. Compared to eukaryotic model systems, bacterial assays are fast and cheap, but cannot detect mutagenesis in targets not conserved between prokaryotes and eukaryotes (such as cytoskeleton, nucleotide excision repair targets). Bacterial assays also cannot detect bioactivation as accurately. Still, direct mutagenesis assays in bacteria constitute one of three assays required by regulatory agencies for the demonstration of safety for potential clinical compounds. The other two assays required are related to a eukaryotic cell culture test and an animal test.
(11) General approaches for detecting mutagenesis in prokaryotes include reporter inactivation (measured in a forward mutation assay) and reversion of an inactivating mutation (measured in a reversion mutation assay). Both the forward mutation assay and the reversion mutation assay are labor-intensive, involving visual screening, quantification of colonies on solid media, and/or obtaining a Poisson distribution in liquid culture. Forward mutation assays are based on the inactivation of a reporter. Reporters can produce colorimetric (e.g., galK, lacZ, luciferase), luminescent, fluorescent (e.g., GFP), or electrochemical signals. Inactivation can result from a variety of mutations. Thus, compared to reversion assays, forward mutation assays detect events that are more frequent. Forward mutation assays also provide a more accurate representation of the range of genetic changes induced by the relevant mutagen because inactivation is generally not dependent on a specific mutation occurring. In some cases, the readout for these involves a survival marker, e.g., a gene that confers resistance to a drug or to the presence or absence of a nutrient. RpoB (a gene encoding for RNA polymerase) is an example, as mutations in a variety of loci produce resistance to rifampin. AraD is another example. The cells used in this assay have a mutation in the araD gene, which results in the accumulation of a toxic intermediate when arabinose is present in the growth media. Mutations upstream of the araD gene that inactivate the operon prevent the metabolism of arabinose, causing the accumulation of the toxic intermediate and making cells resistant to arabinose. Sectoring, which detects inactivation of a reporter within a colony as an indication of high and continuous mutation rates, is another variant of a forward mutation assay. Forward mutation assays are often more labor-intensive because they require screening.
(12) Reversion assays detect when a known inactivating mutation at a pre-determined site is reverted to wild type, typically through a selection (auxotrophy, antibiotic resistance, FACS sorting). The availability of selection increases the sensitivity of these assays relative to the forward mutation assays described above and the reversion assays can also accurately detect a specific mutation. However, the dependence of reversion assays on individual mutations at pre-determined sites makes them susceptible to sequence context effects and limits the range of genetic changes that can be detected. The readout for such an assay can be any of the signals described above (e.g., colorimetric, luminescent, fluorescent, or electrochemical signals). The Ames Test was the first reversion assay to be developed and it is still by far the most widely-used method for testing mutagenesis in prokaryotes. The Ames Test detects the reversion of a mutation that prevents the biosynthesis of histidine and allows the growth of bacteria on solid agar in the presence of trace amounts of histidine. A set of six strains has been developed to detect a broad range of point mutations and frameshift mutations. Two further variations have been developed to facilitate high-throughput formatting and to reduce the amount of sample needed. The Mini-Ames Test follows the standard Ames Test protocol, except at a smaller size. The Ames Fluctuation Test is performed in liquid culture, with the mutation detected by a chromophore that indicates growth. Reversion assays based on TEM β-lactamase have also been developed and one of the assays includes a set of six point mutations reporting on each type of point mutation that is possible in double-stranded DNA. All these reversion assays produce a binary output, i.e., growth vs. no growth. As a result, determining the mutagenicity or genotoxicity of a single concentration of a test compound requires fine-tuning the dose and serial dilutions to obtain countable colonies on solid plates or a sufficient number of positive wells that follow a Poisson distribution in a liquid culture.
(13) Another type of reversion assay is the papillation assay, which can be used to detect alterations in mutagenesis rates in vivo. This assay is based on a mutation in the gal2K gene, which renders cells unable to ferment galactose. Cells are grown on Mac-Conkey-galactose plates, producing white colonies. Spottings on the surface of these colonies, often referred to as colored papilla (sectors), represent microcolonies derived from a single Gal.sup.+ mutant capable of galactose fermentation. The output is only semi-quantitative as it depends on mutation events occurring early enough to allow for visual detection.
(14) Mutator strains are bacterial strains that consistently exhibit an elevated mutation frequency and can be identified by their ability to produce sectored colonies. There are some indications that the mutation rates in mutator strains are not constant, as there is a counter-selection against high mutation rates due to the deleterious and/or adaptive effects of mutations. In addition, studying the dynamics of mutagenesis in mutator strains using reporters is difficult because mutations can inactivate the reporter regardless of its forward or reversion status with a probability that grows exponentially with the number of mutations present.
(15) II. Revision Reporters
(16) Disclosed are reporter systems that can be used to detect and quantify point mutations. These reporters are provided on a plasmid comprising a pMB1 (ColE1-like) plasmid origin of replication. This has several advantages over a chromosomal location: (1) a plasmid reporter increases the number of targets for mutagenesis by at least one order of magnitude, since ColE1 plasmids are multicopy plasmids; (2) the fact that plasmids are present in multiple copies also allows amplification of reporter signal through selection; and (3) a plasmid reporter facilitates exposure to mutagens ex vivo, in this scenario, transformation would be performed only to obtain a readout.
(17) TEM-1
(18) TEM-1 is a type of β-lactamase found in Gram-negative bacteria. Expression of TEM-1 confers resistance to carbenicillin, as well as other β-lactam antibiotics such as penicillin. The version of TEM-1 used the reporters as disclosed herein can be inactivated through mutations in the S68 position of the protein. S68 is a serine residue that polarizes the carbonyl group of the β-lactam amide bond in the β-lactam ring of β-lactamase antibiotics and is completely intolerant to amino acid changes. A set of six TEM-1 constructs with nucleotide point mutations at S68 was engineered such that each nucleotide point mutation is within the serine-coding codon (nucleotides 202 to 204 (“AGC”) of SEQ ID NO: 1 encodes for serine). As a result, each of the 6 pairs of nucleotide substitutions that are possible in duplex DNA can be detected. Point mutations at this position were engineered to be one nucleotide away from a serine-coding codon so that each of the 6 pairs of nucleotide substitutions that are possible in duplex DNA can be detected. Table 1 below lists the nucleotide sequence of wild-type TEM-1 (SEQ ID NO: 1), the nucleotide sequences encoding the six TEM-1 constructs with mutations at S68 codon (SEQ ID NOS: 2-7), and the protein sequence of wild-type TEM-1 (SEQ ID NO: 8).
(19) TABLE-US-00001 TABLE 1 SEQ ID NO: 1 (nucleotide sequence encoding wild-type TEM-1) 1 ATGAGTATTC AACATTTCCG TGTCGCCCTT ATTCCCTTTT TTGCGGCATT TTGCCTTCCT 61 GTTTTTGCTC ACCCAGAAAC GCTGGTGAAA GTAAAAGATG CTGAAGATCA GTTGGGTGCA 121 CGAGTGGGTT ACATCGAACT GGATCTCAAC AGCGGTAAGA TCCTTGAGAG TTTTCGCCCC 181 GAAGAACGTT TTCCAATGAT GAGCACTTTT AAAGTTCTGC TATGTGGCGC GGTATTATCC 241 CGTATTGACG CCGGGCAAGA GCAACTCGGT CGCCGCATAC ACTATTCTCA GAATGACTTG 301 GTTGAGTACT CACCAGTCAC AGAAAAGCAT CTTACGGATG GCATGACAGT AAGAGAATTA 361 TGCAGTGCTG CCATAACCAT GAGTGATAAC ACTGCGGCCA ACTTACTTCT GACAACGATC 421 GGAGGACCGA AGGAGCTAAC CGCTTTTTTG CACAACATGG GGGATCATGT AACTCGCCTT 481 GATCGTTGGG AACCGGAGCT GAATGAAGCC ATACCAAACG ACGAGCGTGA CACCACGATG 541 CCTGTAGCAA TGGCAACAAC GTTGCGCAAA CTATTAACTG GCGAACTACT TACTCTAGCT 601 TCCCGGCAAC AATTAATAGA CTGGATGGAG GCGGATAAAG TTGCAGGACC ACTTCTGCGC 661 TCGGCCCTTC CGGCTGGCTG GTTTATTGCT GATAAATCTG GAGCCGGTGA GCGTGGGTCT 721 CGCGGTATCA TTGCAGCACT GGGGCCAGAT GGTAAGCCCT CCCGTATCGT AGTTATCTAC 781 ACGACGGGGA GTCAGGCAAC TATGGATGAA CGAAATAGAC AGATCGCTGA GATAGGTGCC 841 TCACTGATTA AGCATTGGTA A SEQ ID NO: 2 (nucleotide sequence encoding TEM-1(S68P)) 1 ATGAGTATTC AACATTTCCG TGTCGCCCTT ATTCCCTTTT TTGCGGCATT TTGCCTTCCT 61 GTTTTTGCTC ACCCAGAAAC GCTGGTGAAA GTAAAAGATG CTGAAGATCA GTTGGGTGCA 121 CGAGTGGGTT ACATCGAACT GGATCTCAAC AGCGGTAAGA TCCTTGAGAG TTTTCGCCCC 181 GAAGAACGTT TTCCAATGAT GCCAACTTTT AAAGTTCTGC TATGTGGCGC GGTATTATCC 241 CGTATTGACG CCGGGCAAGA GCAACTCGGT CGCCGCATAC ACTATTCTCA GAATGACTTG 301 GTTGAGTACT CACCAGTCAC AGAAAAGCAT CTTACGGATG GCATGACAGT AAGAGAATTA 361 TGCAGTGCTG CCATAACCAT GAGTGATAAC ACTGCGGCCA ACTTACTTCT GACAACGATC 421 GGAGGACCGA AGGAGCTAAC CGCTTTTTTG CACAACATGG GGGATCATGT AACTCGCCTT 481 GATCGTTGGG AACCGGAGCT GAATGAAGCC ATACCAAACG ACGAGCGTGA CACCACGATG 541 CCTGTAGCAA TGGCAACAAC GTTGCGCAAA CTATTAACTG GCGAACTACT TACTCTAGCT 601 TCCCGGCAAC AATTAATAGA CTGGATGGAG GCGGATAAAG TTGCAGGACC ACTTCTGCGC 661 TCGGCCCTTC CGGCTGGCTG GTTTATTGCT GATAAATCTG GAGCCGGTGA GCGTGGGTCT 721 CGCGGTATCA TTGCAGCACT GGGGCCAGAT GGTAAGCCCT CCCGTATCGT AGTTATCTAC 781 ACGACGGGGA GTCAGGCAAC TATGGATGAA CGAAATAGAC AGATCGCTGA GATAGGTGCC 841 TCACTGATTA AGCATTGGTA A SEQ ID NO: 3 (nucleotide sequence encoding TEM-1(S68T)) 1 ATGAGTATTC AACATTTCCG TGTCGCCCTT ATTCCCTTTT TTGCGGCATT TTGCCTTCCT 61 GTTTTTGCTC ACCCAGAAAC GCTGGTGAAA GTAAAAGATG CTGAAGATCA GTTGGGTGCA 121 CGAGTGGGTT ACATCGAACT GGATCTCAAC AGCGGTAAGA TCCTTGAGAG TTTTCGCCCC 181 GAAGAACGTT TTCCAATGAT GACAACTTTT AAAGTTCTGC TATGTGGCGC GGTATTATCC 241 CGTATTGACG CCGGGCAAGA GCAACTCGGT CGCCGCATAC ACTATTCTCA GAATGACTTG 301 GTTGAGTACT CACCAGTCAC AGAAAAGCAT CTTACGGATG GCATGACAGT AAGAGAATTA 361 TGCAGTGCTG CCATAACCAT GAGTGATAAC ACTGCGGCCA ACTTACTTCT GACAACGATC 421 GGAGGACCGA AGGAGCTAAC CGCTTTTTTG CACAACATGG GGGATCATGT AACTCGCCTT 481 GATCGTTGGG AACCGGAGCT GAATGAAGCC ATACCAAACG ACGAGCGTGA CACCACGATG 541 CCTGTAGCAA TGGCAACAAC GTTGCGCAAA CTATTAACTG GCGAACTACT TACTCTAGCT 601 TCCCGGCAAC AATTAATAGA CTGGATGGAG GCGGATAAAG TTGCAGGACC ACTTCTGCGC 661 TCGGCCCTTC CGGCTGGCTG GTTTATTGCT GATAAATCTG GAGCCGGTGA GCGTGGGTCT 721 CGCGGTATCA TTGCAGCACT GGGGCCAGAT GGTAAGCCCT CCCGTATCGT AGTTATCTAC 781 ACGACGGGGA GTCAGGCAAC TATGGATGAA CGAAATAGAC AGATCGCTGA GATAGGTGCC 841 TCACTGATTA AGCATTGGTA A SEQ ID NO: 4 (nucleotide sequence encoding TEM-1(S68R1)) 1 ATGAGTATTC AACATTTCCG TGTCGCCCTT ATTCCCTTTT TTGCGGCATT TTGCCTTCCT 61 GTTTTTGCTC ACCCAGAAAC GCTGGTGAAA GTAAAAGATG CTGAAGATCA GTTGGGTGCA 121 CGAGTGGGTT ACATCGAACT GGATCTCAAC AGCGGTAAGA TCCTTGAGAG TTTTCGCCCC 181 GAAGAACGTT TTCCAATGAT GAGAACTTTT AAAGTTCTGC TATGTGGCGC GGTATTATCC 241 CGTATTGACG CCGGGCAAGA GCAACTCGGT CGCCGCATAC ACTATTCTCA GAATGACTTG 301 GTTGAGTACT CACCAGTCAC AGAAAAGCAT CTTACGGATG GCATGACAGT AAGAGAATTA 361 TGCAGTGCTG CCATAACCAT GAGTGATAAC ACTGCGGCCA ACTTACTTCT GACAACGATC 421 GGAGGACCGA AGGAGCTAAC CGCTTTTTTG CACAACATGG GGGATCATGT AACTCGCCTT 481 GATCGTTGGG AACCGGAGCT GAATGAAGCC ATACCAAACG ACGAGCGTGA CACCACGATG 541 CCTGTAGCAA TGGCAACAAC GTTGCGCAAA CTATTAACTG GCGAACTACT TACTCTAGCT 601 TCCCGGCAAC AATTAATAGA CTGGATGGAG GCGGATAAAG TTGCAGGACC ACTTCTGCGC 661 TCGGCCCTTC CGGCTGGCTG GTTTATTGCT GATAAATCTG GAGCCGGTGA GCGTGGGTCT 721 CGCGGTATCA TTGCAGCACT GGGGCCAGAT GGTAAGCCCT CCCGTATCGT AGTTATCTAC 781 ACGACGGGGA GTCAGGCAAC TATGGATGAA CGAAATAGAC AGATCGCTGA GATAGGTGCC 841 TCACTGATTA AGCATTGGTA A SEQ ID NO: 5 (nucleotide sequence encoding TEM-1(S68stop)) 1 ATGAGTATTC AACATTTCCG TGTCGCCCTT ATTCCCTTTT TTGCGGCATT TTGCCTTCCT 61 GTTTTTGCTC ACCCAGAAAC GCTGGTGAAA GTAAAAGATG CTGAAGATCA GTTGGGTGCA 121 CGAGTGGGTT ACATCGAACT GGATCTCAAC AGCGGTAAGA TCCTTGAGAG TTTTCGCCCC 181 GAAGAACGTT TTCCAATGAT GTGAACTTTT AAAGTTCTGC TATGTGGCGC GGTATTATCC 241 CGTATTGACG CCGGGCAAGA GCAACTCGGT CGCCGCATAC ACTATTCTCA GAATGACTTG 301 GTTGAGTACT CACCAGTCAC AGAAAAGCAT CTTACGGATG GCATGACAGT AAGAGAATTA 361 TGCAGTGCTG CCATAACCAT GAGTGATAAC ACTGCGGCCA ACTTACTTCT GACAACGATC 421 GGAGGACCGA AGGAGCTAAC CGCTTTTTTG CACAACATGG GGGATCATGT AACTCGCCTT 481 GATCGTTGGG AACCGGAGCT GAATGAAGCC ATACCAAACG ACGAGCGTGA CACCACGATG 541 CCTGTAGCAA TGGCAACAAC GTTGCGCAAA CTATTAACTG GCGAACTACT TACTCTAGCT 601 TCCCGGCAAC AATTAATAGA CTGGATGGAG GCGGATAAAG TTGCAGGACC ACTTCTGCGC 661 TCGGCCCTTC CGGCTGGCTG GTTTATTGCT GATAAATCTG GAGCCGGTGA GCGTGGGTCT 721 CGCGGTATCA TTGCAGCACT GGGGCCAGAT GGTAAGCCCT CCCGTATCGT AGTTATCTAC 781 ACGACGGGGA GTCAGGCAAC TATGGATGAA CGAAATAGAC AGATCGCTGA GATAGGTGCC 841 TCACTGATTA AGCATTGGTA A SEQ ID NO: 6 (nucleotide sequence encoding TEM-1(S68N)) 1 ATGAGTATTC AACATTTCCG TGTCGCCCTT ATTCCCTTTT TTGCGGCATT TTGCCTTCCT 61 GTTTTTGCTC ACCCAGAAAC GCTGGTGAAA GTAAAAGATG CTGAAGATCA GTTGGGTGCA 121 CGAGTGGGTT ACATCGAACT GGATCTCAAC AGCGGTAAGA TCCTTGAGAG TTTTCGCCCC 181 GAAGAACGTT TTCCAATGAT GAACACTTTT AAAGTTCTGC TATGTGGCGC GGTATTATCC 241 CGTATTGACG CCGGGCAAGA GCAACTCGGT CGCCGCATAC ACTATTCTCA GAATGACTTG 301 GTTGAGTACT CACCAGTCAC AGAAAAGCAT CTTACGGATG GCATGACAGT AAGAGAATTA 361 TGCAGTGCTG CCATAACCAT GAGTGATAAC ACTGCGGCCA ACTTACTTCT GACAACGATC 421 GGAGGACCGA AGGAGCTAAC CGCTTTTTTG CACAACATGG GGGATCATGT AACTCGCCTT 481 GATCGTTGGG AACCGGAGCT GAATGAAGCC ATACCAAACG ACGAGCGTGA CACCACGATG 541 CCTGTAGCAA TGGCAACAAC GTTGCGCAAA CTATTAACTG GCGAACTACT TACTCTAGCT 601 TCCCGGCAAC AATTAATAGA CTGGATGGAG GCGGATAAAG TTGCAGGACC ACTTCTGCGC 661 TCGGCCCTTC CGGCTGGCTG GTTTATTGCT GATAAATCTG GAGCCGGTGA GCGTGGGTCT 721 CGCGGTATCA TTGCAGCACT GGGGCCAGAT GGTAAGCCCT CCCGTATCGT AGTTATCTAC 781 ACGACGGGGA GTCAGGCAAC TATGGATGAA CGAAATAGAC AGATCGCTGA GATAGGTGCC 841 TCACTGATTA AGCATTGGTA A SEQ ID NO: 7 (nucleotide sequence encoding TEM-1(S68R2)) 1 ATGAGTATTC AACATTTCCG TGTCGCCCTT ATTCCCTTTT TTGCGGCATT TTGCCTTCCT 61 GTTTTTGCTC ACCCAGAAAC GCTGGTGAAA GTAAAAGATG CTGAAGATCA GTTGGGTGCA 121 CGAGTGGGTT ACATCGAACT GGATCTCAAC AGCGGTAAGA TCCTTGAGAG TTTTCGCCCC 181 GAAGAACGTT TTCCAATGAT GCGCACTTTT AAAGTTCTGC TATGTGGCGC GGTATTATCC 241 CGTATTGACG CCGGGCAAGA GCAACTCGGT CGCCGCATAC ACTATTCTCA GAATGACTTG 301 GTTGAGTACT CACCAGTCAC AGAAAAGCAT CTTACGGATG GCATGACAGT AAGAGAATTA 361 TGCAGTGCTG CCATAACCAT GAGTGATAAC ACTGCGGCCA ACTTACTTCT GACAACGATC 421 GGAGGACCGA AGGAGCTAAC CGCTTTTTTG CACAACATGG GGGATCATGT AACTCGCCTT 481 GATCGTTGGG AACCGGAGCT GAATGAAGCC ATACCAAACG ACGAGCGTGA CACCACGATG 541 CCTGTAGCAA TGGCAACAAC GTTGCGCAAA CTATTAACTG GCGAACTACT TACTCTAGCT 601 TCCCGGCAAC AATTAATAGA CTGGATGGAG GCGGATAAAG TTGCAGGACC ACTTCTGCGC 661 TCGGCCCTTC CGGCTGGCTG GTTTATTGCT GATAAATCTG GAGCCGGTGA GCGTGGGTCT 721 CGCGGTATCA TTGCAGCACT GGGGCCAGAT GGTAAGCCCT CCCGTATCGT AGTTATCTAC 781 ACGACGGGGA GTCAGGCAAC TATGGATGAA CGAAATAGAC AGATCGCTGA GATAGGTGCC 841 TCACTGATTA AGCATTGGTA A SEQ ID NO: 8 (protein sequence of wild-type TEM-1) 1 MSIQHFRVAL IPFFAAFCLP VFAHPETLVK VKDAEDQLGA RVGYIELDLN SGKILESFRP 61 EERFPMMSTF KVLLCGAVLS RIDAGQEQLG RRIHYSQNDL VEYSPVTEKH LTDGMTVREL 121 CSAAITMSDN TAANLLLTTI GGPKELTAFL HNMGDHVTRL DRWEPELNEA IPNDERDTTM 181 PVAMATTLRK LLTGELLTLA SRQQLIDWME ADKVAGPLLR SALPAGWFIA DKSGAGERGS 241 RGIIAALGPD GKPSRIVVIY TTGSQATMDE RNRQIAEIGA SLIKHW (bold nucleotides in SEQ ID NOS: 1-7 correspond to the codon coding for the amino acid at position 68)
(20) The six TEM-1 constructs with mutations at S68 codon (SEQ ID NOS: 2-7) detect the following mutations: SEQ ID NO: 2 (TEM-1 (S68P)) detects C:G.fwdarw.T:A mutations; SEQ ID NO: 3 (TEM-1 (S68T)) detects A:T.fwdarw.T:A mutations; SEQ ID NO: 4 (TEM-1 (S68R1)) detects A:T.fwdarw.C:G and A:T.fwdarw.T:A mutations; SEQ ID NO: 5 (TEM-1 (S68stop)) detects G:C.fwdarw.C:G mutations; SEQ ID NO: 6 (TEM-1 (S68N)) detects A:T.fwdarw.G:C mutations; and SEQ ID NO: 7 (TEM-1 (S68R2)) detects C:G.fwdarw.A:T mutations.
(21) The sequences of the oligonucleotide primers used to introduce the nucleotide mutations at the S68 position of TEM-1 are shown in Table 2.
(22) TABLE-US-00002 TABLE 2 Forward Reverse Mutation Primer T.sub.M Primer T.sub.M S68P TTTCCAAT 54.6° C. ACTCACGT 58.3° C. GATGCCAAC TAAGGGATT TTTTAAAGTT TTGGTCATGA (SEQ ID (SEQ ID NO: 13) NO: 14) S68T TTCCAATG 51.6° C. ACTCACGT 58.3° C. ATGACAAC TAAGGGATT TTTTAAAGT TTGGTCATGA (SEQ ID (SEQ ID NO: 15) NO: 16) S68R1 CCAATGAT 46.3° C. ACTCACGT 58.3° C. GAGAACT TAAGGGATT TTTAAA TTGGTCATGA (SEQ ID (SEQ ID NO: 17) NO: 18) S68stop TTCCAATG 51.6° C. ACTCACGT 58.3° C. ATGTGAAC TAAGGGATT TTTTAAAGT TTGGTCATGA (SEQ ID (SEQ ID NO: 19) NO: 20) S68N CCAATGAT 46.8° C. ACTCACGT 58.3° C. GAACACTTT TAAGGGATT TAAA TTGGTCATGA (SEQ ID (SEQ ID NO: 21) NO: 22) S68R2 CCAATGAT 52.2° C. ACTCACGT 58.3° C. GCGCACTTT TAAGGGATT TAAA TTGGTCATGA (SEQ ID (SEQ ID NO: 23) NO: 24) Q183R GCAGACCA 57.0° C. CGGAAATG 56.5° C. TTATCGACA TTGAATACTC AAATACTCCA ATACTCTTCCT (SEQ ID (SEQ ID NO: 25) NO: 26) (bold nucleotides correspond to the codon coding for the amino acid at position 68)
(23) TEMrev-GFP
(24) The TEMrev-GFP reversion reporter comprises a TEM-1 gene (e.g., SEQ ID NO: 1) or a mutant thereof (e.g., any one of SEQ ID NOS: 2-7) positioned 5′ to a GFP or a GFP derivative in an expression plasmid. In some embodiments, a linker may be placed between the TEM-1 and the GFP. Examples of linkers are described in detail further herein. One example of a GFP derivative is Cycle 3 GFP, a variant of GFP optimized for fluorescence in E. coli. Other fluorescent proteins that may be used in the reporters and methods of the disclosure are described in detail further herein. The TEM-1 gene (e.g., SEQ ID NO: 1) or the mutant thereof (e.g., any one of SEQ ID NOS: 2-7) may be placed in the same plasmid and co-transcribed with the GFP or GFP derivative (
(25) sfGFPrev-TEM
(26) The sfGFPrev-TEM reporter comprises an inactivated GFP derivative (superfold GFP (sfGFP)) and a TEM-1 (e.g., SEQ ID NO: 1). The inactivated GFP derivative (sfGFPrev) comprises a stop codon in the GFP, rending it non-fluorescent. In some embodiments, a linker may be placed between the sfGFPrev and TEM-1 (e.g., a 12-amino acid serine and glycine-rich linker) (
(27) TABLE-US-00003 SEQ ID NO: 12 (squiggly: sfGFP (SEQ ID NO: 10); bold: linker; underlined: TEM-1)
(28) Superfolder GFP (sfGFP) is a derivative of Cycle 3 GFP that includes two additional mutations selected for robustness to translational fusions. sfGFPrev is preferably located at the N-terminus of TEM-1 and is preferably expressed in Top10 cells rather than JS200, AB 1157, or GW7101. The introduction of a stop codon truncating GFP expression inactivates both GFP and TEM-1, since TEM-1 is placed downstream of sfGFP, resulting in a non-fluorescent, carbenicillin-sensitive phenotype. A TGA stop codon may be introduced into sfGFP at three different positions: Q69, K113, and K126 (see SEQ ID NO: 10). Reversion of the stop codon in the mutated sfGFP back to the codon encoding the original amino acid at the particular position (e.g., Q at position 69, K at position 113, and K at position 126 of SEQ ID NO: 10) may result in fluorescence and successful translation of the entire sfGFPrev-TEM reporter. Thus, the translated and functional TEM-1 may be able to provide carbenicillin resistance to the cells.
(29) TEMrev-GFPrev
(30) The TEMrev-GFPrev reporter monitors mutagenesis in mutator strains quantitatively. TEMrev-GFPrev comprises a Cycle 3 GFP inactivated via a Q183R (CAA to CGA) mutation, which reverts to functional Cycle 3 GFP in response to a C:G.fwdarw.T:A mutation, and a TEM-1 mutant (e.g., any one of SEQ ID NOS: 2-7). Thus, this reporter couples two reversion assays: TEM-1 reversion and GFP reversion. This double set of markers allows the detection of sequential hits, separating beyond double mutation events in time and facilitating the detection of changes in mutation rates over time. The TEM-1 and the GFP may be included in the same plasmid and their expression can be driven by the same or different promoters. In some embodiments, a linker may be placed between the TEM-1 and the GFP. An example of a method of using the reporter is shown schematically in FIGS. 2A and 2B. Colonies containing the plasmid are plated on carbenicillin plates to identify reversion events in TEM-1 (i.e., a mutant TEM-1 that is inactive (any one of SEQ ID NOS: 2-7) being reverted back to the wild-type and active TEM-1 (SEQ ID NO: 1)). Non-fluorescent colonies (i.e., the colonies containing wild-type and active TEM-1 and inactivated GFP) are picked and grown in liquid culture. The plasmid DNA from these cultures is recovered and retransformed into a readout strain (e.g., Top10 or DH5α) to identify reversion events in GFP (e.g., non-fluorescent and inactivated GFP containing R at position 183 being reverted back to the fluorescent and active GFP containing Q at position 183) (
(31) The TEMrev-GFPrev reporter presents an alternative to papillation assays for the characterization of mutator strains. The main advantage is that the output in this case is quantitative rather than semi-quantitative, allowing head-to-head comparisons between different mutators and/or growth conditions. Different inactivating GFP mutations can be introduced, depending on the mutagenic profile of the mutator strain. The chromophore-containing cyclized hexapeptide (amino acids at positions 64 to 69) is a good target for an inactivating mutation with a narrow tolerance to alternative amino acids. Other possible mutations in Cycle 3 GFP are described further herein (see, e.g., Table 4).
(32) III. Fluorescent and Non-Fluorescent Proteins
(33) The three reporters described above (TEMrev-GFP, sfGFPrev-TEM, and TEMrev-GFPrev) utilize a GFP or a derivative thereof in order to obtain a fluorescence readout of the reversion event. GFP facilitates quantification of growth in vivo, producing a much stronger signal than turbidity. Note that in
(34) The sequences of Cycle 3 GFP (SEQ ID NO: 9) and superfold GFP (sfGFP) (SEQ ID NO: 10) are shown in Table 3 below. The bold amino acids in SEQ ID NO: 9 represent the 12 amino acids each of which may be mutated to inactivate Cycle 3 GFP (Table 4 below further describes specific mutated nucleotides). The bold amino acids in SEQ ID NO: 10 (Q69, K113, and K126) are the amino acids each of which may be mutated to a stop codon (e.g., TGA) to create an inactive and truncated sfGFP.
(35) TABLE-US-00004 TABLE 3 SEQ ID NO: 9 (protein sequence of Cycle 3 GFP) MSKGEELFTGVVPILVELVDGDVNGHKFSVSGEGEGDATYGKLTLKF ICTTGKLPVPWPTLVTTFSYGVQCFSRYPDHMKRHDFFKSAMPEGYV QERTISFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHK LEYNYNSHNVYITADKQKNGIKANFKIRHNIEDGSVQLADHYQQNTP IGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITHGMD ELYK SEQ ID NO: 10 (protein sequence of sfGFP) MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFI CTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQ ERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKL EYNFNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPI GDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEFVTAAGITHGMDE LYK
(36) Other fluorescent proteins may also be used in the reporters described herein. Examples of fluorescent proteins are well-known in the art, see, e.g., Gert-Jan Kremers et al., J Cell Sci. 124:157, 2011 and Stepanenko et al., Curr Protein Pept Sci. 9:338, 2008. Examples of fluorescent proteins include, but are not limited to, green fluorescent protein (GFP), yellow fluorescent protein (YFP), enhanced blue fluorescent protein (EBFP), azurite, GFPuv, T-Sapphire, Cerulean, mCFP, mTurquoise2, ECFP, CyPet, mKeima-Red, TagCFP, AmCyan1, mTFP1, Midoriishi Cyan, TurboGFP, TagGFP, Emerald, Azami Green, ZsGreen1, TagYFP, EYFP, Topaz, Venus, mCitrine, YPet, TurboYFP, ZsYellow1, Kusabira Orange, mOrange, Allophycocyanin (APC), mKO, TurboRFP, tdTomato, TagRFP, DsRed monomer, DsRed2, mStrawberry, TurboFP602, AsRed2, mRFP1, J-Red, R-phycoerythrin (RPE), B-phycoerythrin (BPE), mCherry, HcRedl, Katusha, P3, Peridinin Chlorophyll (PerCP), mKate (TagFP635), TurboFP635, mPlum, and mRaspberry.
(37) To create an inactive or non-fluorescent version of a fluorescent protein, in some embodiments, a stop codon may be introduced within the protein sequence. For example, the bold amino acids in SEQ ID NO: 10 (Q69, K113, and K126) indicate the amino acids each of which may be mutated to a stop codon (e.g., TGA) to create an inactive and truncated sfGFP. Table 4 further lists the potential mutants and the mutated nucleotide that may be introduced into Cycle 3 GFP to create a non-fluorescent Cycle 3 GFP. A sequence of a non-fluorescent Cycle 3 GFP is shown in SEQ ID NO: 11, wherein Q at position 183 of SEQ ID NO: 9 is mutated to an R. Further, as described above, one or more amino acids within the chromophore-containing cyclized hexapeptide of a fluorescent protein (i.e., amino acids at positions 64 to 69) may be mutated to produce a non-fluorescent protein. One of skill in the art would have the ability and knowledge to identify amino acids within the chromophore of a fluorescent protein, see, e.g., Stepanenko et al., Biotechniques 51(5): 313, 2011, Sarkisyan et al., Sci Reports 2:608, 2012, and Gross et al., Proc Natl Acad Sci USA. 97(22): 11990-11995, 2000.
(38) TABLE-US-00005 SEQ ID NO: 11 (Cycle 3 GFP (Q183R)): MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTT GKLPVPWPTLVTTFSYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISF KDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNV YITADKQKNGIKANFKIRHNIEDGSVQLADHYRQNTPIGDGPVLLPDNHY LSTQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYK
(39) TABLE-US-00006 TABLE 4 Amino acid Nucleotide Original Mutant position of substi- Original amino amino SEQ ID NO: 9 tution Codon acid acid 48 G to A TGC C Y 49 A to G ACT T A 56 C to T CCA P L 61 T to A GTC V D 62 A to G ACT T A 89 C to T CCC P S 91 G to A GGT G D 92 A to G TAT Y C 103 A to T GAC D V 143 A to G TAC Y C 183 A to C CAA Q H 213 A to G GAA E G
(40) In some embodiments, a reporter may contain a mutant TEM-1 (e.g., the sequence of any one of SEQ ID NOS: 2-7) joined to the N-terminus of a fluorescent protein (similar to the TEMrev-GFP reporter described above). A reporter may also contain a mutant and inactive fluorescent protein joined to the N-terminus of a wild-type TEM-1 (e.g., the sequence of SEQ ID NO: 1) (similar to the sfGFPrev-TEM reporter described above). In some embodiments, a reporter may contain a mutant TEM-1 (e.g., the sequence of any one of SEQ ID NOS: 2-7) joined to the N-terminus or C-terminus of a mutant and inactive fluorescent protein (similar to the TEMrev-GFPrev reporter described above). In any of the reporters described herein, a linker may be optionally placed between the TEM-1 or a mutant thereof and the active or inactive fluorescent protein. Examples of linkers are described in detail further herein.
(41) IV. Linkers
(42) In some embodiments, a linker may be used as a linkage or connection between a TEM-1 or a mutant thereof and an active or inactive fluorescent protein. The linker may be a peptide including, e.g., 3-200 amino acids (e.g., 3-200, 3-180, 3-160, 3-140, 3-120, 3-100, 3-90, 3-80, 3-70, 3-60, 3-50, 3-45, 3-40, 3-35, 3-30, 3-25, 3-20, 3-15, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-200, 5-200, 6-200, 7-200, 8-200, 9-200, 10-200, 15-200, 20-200, 25-200, 30-200, 35-200, 40-200, 45-200, 50-200, 60-200, 70-200, 80-200, 90-200, 100-200, 120-200, 140-200, 160-200, or 180-200 amino acids). In some embodiments, a linker may be a peptide including 8-16 amino acids (e.g., 8, 9, 10, 11, 12, 13, 14, 15, or 15 amino acids). Suitable linkers are known in the art, and include, for example, peptide linkers containing flexible amino acid residues such as glycine and serine. In certain embodiments, a linker can contain motifs, e.g., multiple or repeating motifs, of GS, GGS, GGGGS (SEQ ID NO: 27), GGSG (SEQ ID NO: 28), or SGGG (SEQ ID NO: 29). In certain embodiments, a linker can contain 2 to 12 amino acids including motifs of GS, e.g., GS, GSGS (SEQ ID NO: 30), GSGSGS (SEQ ID NO: 31), GSGSGSGS (SEQ ID NO: 32), GSGSGSGSGS (SEQ ID NO: 33), or GSGSGSGSGSGS (SEQ ID NO: 34). In certain other embodiments, a linker can contain 3 to 12 amino acids including motifs of GGS, e.g., GGS, GGSGGS (SEQ ID NO: 35), GGSGGSGGS (SEQ ID NO: 36), and GGSGGSGGSGGS (SEQ ID NO: 37). In yet other embodiments, a linker can contain 4 to 20 amino acids including motifs of GGSG (SEQ ID NO: 28), e.g., GGSGGGSG (SEQ ID NO: 38), GGSGGGSGGGSG (SEQ ID NO: 39), GGSGGGSGGGSGGGSG (SEQ ID NO: 40), or GGSGGGSGGGSGGGSGGGSG (SEQ ID NO: 41). In other embodiments, a linker can contain motifs of GGGGS (SEQ ID NO: 27), e.g., GGGGSGGGGS (SEQ ID NO: 42) or GGGGSGGGGSGGGGS (SEQ ID NO: 43).
(43) In other embodiments, a linker can also contain amino acids other than glycine and serine, e.g., GSAGSAAGSGEF (SEQ ID NO: 44), GENLYFQSGG (SEQ ID NO: 45), or SACYCELS (SEQ ID NO: 46). In particular embodiments, the linker is GSAGSAAGSGEF (SEQ ID NO: 44).
EXAMPLES
Example 1—Brief Description of Experimental Protocol
(44) Materials
(45) Transformation and Mutagenesis
(46) Competent cells: Top10, JS200-pHSG_WTPolA, and JS200-pHSG_EP1 PoIA
(47) ColE1 vectors: pGFPck (Cycle 3; fluorescent), pGFPck (Q183R; non-fluorescent), sfGFP, and pGFPuv_KanR
(48) Other materials: 500 mL centrifuge bottles, Eppendorf centrifuge 5810 R (Eppendorf), 50 mL conical tubes (Fisher Scientific, Cat.#1443222), 15 mL culture tubes (E&K Scientific, Cat.# EK-62262), LB broth (Fisher Scientific, Cat.# BP1426-2), LB agar (Fisher Scientific, Cat.# BP1425-2), 100 mm×15 mm disposable Petri dishes (Fisher Scientific, Cat.# FB0875713), kanamycin solution (30 mg/mL, store at −20° C.), kanamycin (30 μg/mL) LB agar and broth, 1.5 mL microfuge tubes (E&K Scientific, Cat.#280150), TropiCooler, Model 260014 (Boekel Scientific), MaxQ 4000 shaker/incubator (Barnstead International), and water-jacketed incubator (Forma Scientific)
(49) Washing Plates
(50) Materials: kanamycin (30 μg/mL) LB broth, plate spinner, plate spreader, ethanol (200 proof), Bunsen burner, spectrophotometer cuvettes (Fisher Scientific, Cat.#14955127), 1.5 mL microfuge tubes (E&K Scientific, Cat.#280150), and BioMate 3 Spectrophotometer (Thermo Scientific)
(51) Readout (Plates)
(52) Materials: kanamycin (30 μg/mL) LB agar and broth, carbenicillin (100 pg/mL) LB agar and broth, 1.5 mL microfuge tubes (E&K Scientific, Cat.#280150), plate spreader, plate spinner, ethanol (200 proof), Bunsen burner, water-jacketed incubator (Forma Scientific), and UV Light
(53) Readout (Liquid Culture Assay)
(54) Materials: kanamycin (30 μg/mL) LB broth, carbenicillin (100 μg/mL) LB broth, 3 mm diameter glass beads (Sigma-Aldrich, Cat.# Z265926), AirPore tape sheets (Qiagen, Cat.#2017-10-RP), 96-well round-bottomed deep-well plates (Fisher Scientific, Cat.#10011-944), 96-well flat-bottomed black-walled plates (Fisher Scientific, Cat.#82050-744), microtiter plate lids (Fisher Scientific, Cat.#82050-829), MaxQ 4000 shaker/incubator (Barnstead International), SpectraMax M2e Fluorometric and Spectrophotometric plate reader, dual monochromators, and Absorbance 200-1000 nm and excitation 250-850 nm (Molecular Devices)
(55) Plasmid Recovery
(56) Materials 15 mL culture tubes (E&K Scientific, Cat.# EK-62262), 1.5 mL microfuge tubes (E&K Scientific, Cat.#280150), and Nucleospin Plasmid (NoLid) kit (Macherey-Nagel, Cat.#740499.250)
(57) Sequencing Plasmids of Interest
(58) NanoDrop ND-1000 Spectrophotometer for DNA quantification (Thermo Scientific), 0.6 mL microfuge tubes (E&K Scientific, Cat.#280060-S), and MacVector version 12.7.5 for sequence analysis (MacVector Inc.)
(59) Methods
(60) Transformation of ColE1 Plasmids by Heat-Shock Method
(61) Prepare a 5 mL overnight culture in a 15 mL culture tube in LB media for the cell line of interest. If necessary, include selective antibiotic in the media for the desired cell line. Expand this culture into a sterile 1 L Erlenmeyer flask containing 500 mL of LB media with selective antibiotic. Incubate this flask at 30° C. or 37° C. (depending on the cell line; incubate JS200 cell lines at 30° C., and all others at 37° C.) with shaking (225 rpm) until exponential phase is reached (OD.sub.600=0.4-0.6). Chill the flask containing the cells on ice for 20 minutes. For best results, cells should be kept chilled at all times. Transfer the liquid cultures to 500 mL plastic centrifuge bottles and centrifuge at 4000 rpm for 20 minutes at 4° C. Pour off supernatant, and resuspend the cell pellet in 50 mL of chilled calcium chloride solution (100 mM CaCl.sub.2, 10 mM HEPES, 15% Glycerol, pH 7). Transfer the resuspended cells into a 50 mL conical tube. Centrifuge the cells at 4000 rpm for 20 minutes at 4° C. Pour off supernatant, and resuspend the cell pellet in 50 mL of chilled calcium chloride solution, and centrifuge the cells at 4000 rpm for 20 minutes at 4° C. (repeat 3 times).
(62) After third wash with calcium chloride solution, pour off supernatant, and resuspend the cells in 5 mL of chilled calcium chloride solution. Keep cells on wet ice and use immediately, or aliquot into 1.5 mL microfuge tubes and place on dry ice for storage at −80° C. Pipette 40 μL of cells into 1.5 mL microfuge tube per transformation. Pipette 100 μg of plasmid DNA into the tube containing the competent cells and mix well by pipetting up and down. Incubate on ice for 30 minutes. Heat-shock the cells at 42° C. for 90 seconds on Tropicooler block. Place cells back on ice for 5 minutes. Add 1 mL of LB Broth to the microfuge tube containing the cells and DNA. Allow the cells to recover for 30 minutes to 1 hour at 30° C. or 37° C. (depending on the cell line; incubate JS200 cell lines at 30° C., and all others at 37° C.) with shaking (225 rpm). Plate transformed cells by spreading 100 μL with sterile plate spreader onto pre-warmed LB agar plates containing 30 μg/mL kanamycin. Allow the cells to grow overnight at either 30° C. or 37° C.
(63) Inducing Mutagenesis
(64) For ColE1 on plasmids which have been transformed into JS200-pHSG_EP1 PoIA cell strains, incubate at 37° C. to induce mutagenesis. Use the same plasmids transformed into JS200-pHSG_WTPolA as control.
(65) Washing Plates
(66) Observe plate for bacterial colony lawn formation, which consists of a high density of colonies. Place plate on a plate spinner. Add 1 mL LB broth containing 30 μg/mL kanamycin directly to the plate surface containing bacterial growth. Use sterile plate spreader to collect colonies into LB Broth. Tilt plate slightly of collect broth containing harvested colonies into one area, and transfer as much as possible into a 1.5 mL microfuge tube. Repeat colony harvesting steps again. Collect second wash into the same 1.5 mL microfuge tube. Dilute plate washes 1:20 directly in spectrophotometer cuvettes (950 μL media+50 μL plate wash), and mix by pipetting. Measure OD.sub.600 of diluted plate wash using the BioMate 3 spectrophotometer, and multiply the measurement by 20 to obtain the actual OD.sub.600 of the undiluted plate wash. Normalize all plate washes to OD.sub.600=1 prior to readout experiments.
(67) Readout (Plates)
(68) Pre-warm LB agar plates containing 30 rig/mL kanamycin and LB agar plates containing 100 μg/mL carbenicillin in incubator set at 30° C. or 37° C. (depending on the cell line; incubate JS200 cell lines at 30° C., and all others at 37° C.). Plate 100 μL of plate washes to pre-warmed plates (all washes plated to both LB agar plates containing 30 μg/mL kanamycin and LB agar plates containing 100 μg/mL carbenicillin) at appropriate dilutions to yield countable colonies. Incubate at 30° C. or 37° C. (depending on the cell line; incubate JS200 cell lines at 30° C., and all others at 37° C.) overnight. Determine the number of colonies on each plate. Use counts to determine CFU/mL of OD normalized cultures on each type of selective media.
(69) Determine percent of TEM β-lactamase S68 revertants by the formula:
% Reversion=[(CFU/mL(carbenicillin))/(CFU/mL(kanamycin))]*100
(70) Readout (Liquid Culture Assay)
(71) Aseptically place one sterile 3 mm diameter glass bead into each well of a 96-well deep-well round-bottomed plate using sterilized forceps. Transfer 950 μL of LB broth containing 30 pg/mL kanamycin to one well and 950 μL of LB broth containing 100 μg/mL carbenicillin to another well for each construct to be tested at each time point. Inoculate wells with 50 μL of 1:10 diluted plate washes (final inoculation OD.sub.600=0.05). Cover with AirPore tape sheet. Remove 200 μL of TO time point to 96-well black-walled clear-bottomed flat-bottomed plates, cover with sterile plate lid, place at 4° C. Cover deep-welled plate with sterile plate lid and place in incubator at 30° C. or 37° C. (depending on the cell line; incubate JS200 cell lines at 30° C., and all others at 37° C.) with shaking (325 rpm). At appropriate time points, remove 200 μL of culture from pre-assigned well to 96-well black-walled plates. Between time points, the black-walled plates should be stored at 4° C., and the deep-welled plates should be incubated at the appropriate temperature with shaking. At the last time point, remove culture and un-inoculated blank wells to black-walled plates. Read OD.sub.600 and fluorescence (ex. 395 nm, em. 509 nm) from black-walled plates on SpectraMax M2e Fluorometric and Spectrophotometric plate reader. Plot OD.sub.600 versus time and fluorescence versus time for constructs under both kanamycin selection and carbenicillin selection to estimate relative rates of TEM β-lactamase S68 reversion.
(72) Plasmid Recovery
(73) Pick reversion colonies from LB agar plates containing 100 μg/mL carbenicillin generated previously, and inoculate into 3 mL of LB broth containing 100 μg/mL carbenicillin. Grow cultures overnight at 30° C. or 37° C. (depending on the cell line; incubate JS200 cell lines at 30° C., and all others at 37° C.) with shaking (225 rpm). Harvest cells by centrifugation at 11,000×g for 1 minute, pour off supernatant, and isolate plasmid DNA (miniprep) based on manufacturer's instructions.
(74) Sequencing Plasmids of Interest
(75) Quantify plasmid DNA yield and purity using NanoDrop Spectrophotometer. Open NanoDrop software (ND-1000, version 3.8.1), and select nucleic acid quantification. Place 2 μL of purified water on cleaned pedestal and lower arm to initialize spectrophotometer. Place 2 μL of elution buffer on cleaned pedestal and lower arm to blank spectrophotometer. Place 2 μL of sample to be quantified on cleaned pedestal and lower arm to measure absorption spectrum between 220 nm and 350 nm. Transfer 0.5 to 1 μg of plasmid DNA to 0.6 μL microfuge tube with appropriate label. Transfer 10 μL of 5 μM sequencing primer to 0.6 μL microfuge tube with appropriate label. Send plasmid DNA and sequencing primer to sequencing facility. Assemble and analyze sequences using the program MacVector version 12.7.5.
Other Experimental Notes
(76) Pre-warming plates prior to plating cells is essential for efficient mutagenesis. Typical recovery per 2 mL of LB is about 1.5 mL of plate wash. Experimenter must estimate dilution needed to achieve a total number of colonies on plate between 30-300. This may require some trial and error. The results indicate that a dilution factor of 10.sup.−7 is effective for all constructs and controls on kanamycin plates and positive controls (WT β-lactamase) on carbenicillin plates and no dilution for negative controls on carbenicillin plates. For reporter constructs on carbenicillin plates, dilutions may vary depending on the expected reversion frequency, but generally range between no dilution and a dilution factor of 10.sup.−3. Be sure to take note of the dilution factor used for each construct plated, as this will be used to calculate CFU/mL.
(77) Sterilize forceps by dipping in ethanol and holding over flame until red hot. It is recommended to have replicates at each time point. Inoculate different time points and different constructs and controls into separate wells. Inoculate each culture into both kanamycin wells and carbenicillin wells. Leave at least three wells on each plate un-inoculated, to be used as blanks during spectrophotometry/fluorimetry. For cell lines growing at 30° C., culture growth will be slower. Plan time points accordingly. Take 200 μL from fresh wells at each time point. Do not resample wells that have already been sampled at previous time points. Sample well by stabbing the micropipette tip through the AirPore sheet. Use caution to avoid disturbing/cross-contaminating wells containing later time points or blanks.
Example 2—Experimental Validation
(78) To validate the reporter system, an error-prone Pol I plasmid replication was used. This system is based on expression of an error-prone variant of DNA polymerase I (Pol I) in JS200, a polAl2 (temperature-sensitive) strain of E. coli. Shift of this strain to 37° C. makes J200 cells dependent on the activity of the error-prone variant of Pol I (low fidelity Pol I or LF-Pol I) for survival. Specifically, LF-Pol I performs ColE1 plasmid replication and processes of Okazaki fragments during lagging-strand replication in both the plasmid and in chromosomal DNA. This variant bears three mutations that decrease its replication fidelity: 1709N in motif A (broadening its active site), A759R in motif B (favoring its closed conformation), and D424A (inactivating its proofreading domain).
(79) Overnight culture under restrictive conditions (37° C.) leads to an increased mutation frequency in ColE1 plasmids by over three orders of magnitude in vivo, about 1 nucleotide substitution per 1.5 kb. This is true for most of the plasmid sequence, where Pol I appears to be competing with Pol III. These loads are higher in areas replicated exclusively by Pol I: the 150 nucleotides immediately downstream of the RNA/DNA switch (leading-strand synthesis by Pol I), about 500 nucleotides upstream of the RNA/DNA switch (gap-filling of lagging-strand synthesis by Pol I), and about 20 nucleotide patches corresponding to areas of Okazaki fragment processing by Pol I. It is worth noting that LF-Pol I is partially dominant in vivo, as expression of this polymerase still produces ColE1 plasmid mutagenesis at permissive temperature or in polA WT strains, albeit with about 4 fold lower frequency relative to JS200 at restrictive temperature.
(80) In terms of mutation spectrum, the mutation frequency of LF Pol I on a single strand in vivo was estimated. The vast majority of mutations (>95%) are point mutations and can be grouped in four groups: most frequent: C.fwdarw.T transitions (60%); frequent: A.fwdarw.G and A.fwdarw.T (20 and 10% of the total), respectively); rare: G.fwdarw.T, G.fwdarw.A, and G.fwdarw.T, and extremely rare: T.fwdarw.C, T.fwdarw.A, A.fwdarw.C, and C.fwdarw.G. The observation of the very low frequency of T.fwdarw.C transitions indicates that mismatch repair appears to be intact in these cells. Given that the reporter detects mutations in double-stranded DNA, i.e., in pairs of complementary mutations, the following ranking based on frequency is expected: C to T/G to A (most frequent)>A to G/T to C, A to T/T to A>G to T/C to A>T to G/A to C>G to C/C to G.
Example 3—Reversion Detection Using Six TEMrev-GFP Reporters
(81) Six TEMrev-GFP reporters (e.g., each of the sequence of any one of SEQ ID NOS: 2-7 joined to the sequence of GFP), a TEM-GFP positive control, and a negative control not bearing the TEM1 gene (
(82) For the TEMrev-GFP reporter, growth of the transformants overnight produced a high density of colonies (near-lawn). These colonies were harvested from the plate into about 1.5 ml of LB broth. Absorbance at 600 nm was determined to normalize the washes to OD.sub.600=1. These dilutions were used to plate kanamycin plates (at further dilution of 1:10.sup.7) and carbenicillin plates at different dilutions, depending on reversion frequencies (between neat and 1:10.sup.3 dilutions). This time plates were incubated overnight at 30° C. to minimize additional mutagenesis. Following incubation, the number of colonies on each plate were counted, and this number was used to calculate the reversion rate for each reporter (
(83) Three of the reporters produced between 10 and 100-fold higher background mutation frequencies in the control strain expressing WT Pol I relative to the other three (
(84) Two pairs exhibit lower reversion frequencies than expected: A:T.fwdarw.G:C and G:C.fwdarw.T:G. This could be the result of sequence-context dependent effects, which can be in part due to differential efficiency of mismatch repair. The observation that S68R1, which detects A:T.fwdarw.C:G and A:T.fwdarw.T:A mutations, produces fewer reversions than S68T, which detects A:T.fwdarw.T:A alone directly confirms the impact of local sequence context on mutation rates. Overall, then, profiling a mutation spectrum using the disclosed TEMrev-GFP reporter gives a general idea of which types of point mutations are favored, particularly if there is as strong bias for a specific type.
(85) For the sfGFPrev-TEM reporter containing K126stop mutation in sfGFPrev, JS200 cells expressing LF-Pol I and transformed with the sfGFPrev-TEM reporter that were plated at 30° C. produced a semi-lawn of carbenicillin-resistant, fluorescent colonies. Sequencing of 10 these colonies showed point mutations at the stop codon in all cases, producing an L (three times), W (three times), Q (twice), and Y (twice). In eight of these cases, the WT signal was still detectable, suggesting that the plasmid carrying the K126 point mutation had not replaced all the copies of the original K126 stop reporter. Cells expressing WT-Pol I had practically no colonies (
Example 4—Growth and Fluorescence Emission Kinetics
(86) Following mutagenesis, plates were washed with LB media and normalized to OD.sub.600=1.0 as described above. These cultures were used to inoculate 96-well plates in a 1:20 dilution. The plates were deep-well round-bottom plates with glass beads (to facilitate oxygenation) and a final volume of 1 mL was added to each well. The plates were then covered with AirPore breathable sheets, in order to protect against cross-contamination and evaporation effects, while still allowing for microbe growth under aerobic conditions. Cells were grown at 30° C. with shaking at 325 rpm. At different time-points, 200 μL of each culture was transferred to a set of black-walled flat-bottomed 96-well microtiter plates and kept at 4° C. At the end of the experiment, these plates were read on a fluorescence-enabled spectrophotometric plate reader for absorbance at 600 nm to determine growth and for fluorescence (with excitement λ=395 nm, and emission λ=509 nm). Results were then used to plot growth kinetics curves for each construct under each antibiotic selection.
Example 5—Analysis of Double Reversion Events
(87) For continuous mutagenesis detection, colonies expressing LF-Pol I and bearing the TEMrev-GFPrev reporter were plated under restrictive conditions as described above, but at a higher dilution factor in order to obtain individual carbenicilin-resistant colonies. Three non-fluorescent colonies were picked, and grown in liquid culture under restrictive conditions. The DNA from these cultures was recovered and retransformed into DH5α cells to identify R183Q (fluorescent) revertants (
(88) A reversion frequency at the Q183R site (revert R183 back to Q183) was found to be about 1 in 10.sup.4 cells. This frequency is 2-3 fold lower than that observed at the TEMrev site (revert a mutant TEM-1 back to a wild-type TEM-1). It is unclear which amino acid substitutions are allowed at this site, but a C:G-T:A mutation reverts R at position 183 back to Q. Given that C:G-T:A mutations are the predominant mutations introduced by LF-Pol I, it can be assumed that this is the main mutation driving the reversion.
(89) To control for the possibility that R183Q reversions were already present in one of the copies of the plasmid pool of the original colony, the original carbenicillin-resistant colonies were expanded under permissive conditions as well. Only 2 fluorescent colonies were observed in 9,300 transformants, and no fluorescent colonies were observed in plasmids recovered from cells expressing WT Pol I (113,000 transformants). Given that the average plasmid copy number for the reporter plasmid in LF-Pol expressing cells is less than ten plasmids per cell, these results confirm that the observed fluorescent colonies are most likely the result of mutations at the 183 position of GFP that occurred after the P68S reversion.
(90) A reversion frequency that is far lower than 1 divided by plasmid copy number confirms that the revertants were not present in the cell where the original reversion of the TEM-1 marker occurred, and therefore argues that the GFP reversion occurred at a later time-point. This observation has two additional implications: (1) it suggests that under restrictive conditions, LF-Pol I-expressing cells continue to generate mutations after a first passage, albeit at a reduced rate relative to early culture; (2) it also suggests that at the permissive temperature, where the mutation rate is already low, mutation rates can be sustained over longer periods of time. Random mutagenesis systems that maintain mutation rates over time would greatly facilitate directed evolution. Thus, the disclosed reporter can be used to fine-tune existing mutator strains such as XL-1 red, the MP6 mutagenesis system, or strains with altered dNTP pools to identify conditions supporting constant mutation rates over time.