WO1993005171A1 - Procede permettant de determiner la sensibilite d'une technique de detection d'adn - Google Patents

Procede permettant de determiner la sensibilite d'une technique de detection d'adn Download PDF

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Publication number
WO1993005171A1
WO1993005171A1 PCT/US1992/006535 US9206535W WO9305171A1 WO 1993005171 A1 WO1993005171 A1 WO 1993005171A1 US 9206535 W US9206535 W US 9206535W WO 9305171 A1 WO9305171 A1 WO 9305171A1
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Prior art keywords
lysogen
gene
interest
bacteria
population
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PCT/US1992/006535
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English (en)
Inventor
Robert A. Lepley
William Gerard Mc Donald
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The Upjohn Company
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Publication of WO1993005171A1 publication Critical patent/WO1993005171A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor

Definitions

  • the present invention relates to an assay to determine the level of sensitivity a DN detection method can achieve, such a DNA detection method being one useful in the detecting th presence of bacteria that carry a particular DNA sequence in a large heterogenous population o bacteria which do not contain the particular DNA sequence.
  • transcriptionally active recombinant DNA into the environment, eithe intentionally or during the manufacture of recombinant proteins, is a safety concern of both th biotechnology industry and government regulatory agencies including the Food and Dru Administration and the Environmental Protection Agency.
  • Capture of transcriptionally active DN by naturally occurring microorganisms could result in replicating bacteria different from th originally engineered strain and capable of producing the recombinant protein which the capture DNA encodes.
  • Bacteria can transfer or exchange genetic material by mating, by infection b bacterial viruses called bacteriophages, or through other means of taking up genetic material fro the environment.
  • the methodology used in these studies generally involve oral exposure to the engineered bacteria followed by screening the bacteria in the fecal material of the animal for bacteria containing the gene of interest.
  • Oral administration corresponds to the likely route bacteria enter the animals body. Bacteria regularly inhabit the intestinal tracts of animals and genetic transfer between engineered and endogenous species or colonization by engineered strains will likely take place there. The examination of microbial flora in fecal matter is a sound method of surveying the inhabitants of an animal's gut.
  • the process of gene detection in heterologous populations can be improved upon by choosing a suitably sensitive detection method and by providing a means to validate the method's limits of sensitivity.
  • One such means of approach to the binary problem of sensitivity and verification is to generate an organism to serve as a model the organism which may result from the transfer of heterologous DNA.
  • Such a model organism could serve as an important assay standard and could be conveniently and expeditiously engineered by the use of known transducing bacteria phages.
  • the use of such an organism coupled with a sensitive gene detection technique, e.g., DNA hybridization, could be used as a validated assay system.
  • data generated by such assays will provide a guide to the accuracy and range of sensitivity of detection assays used to determine the presence and/or movement of genes of interest in bacteria which inhabit animal hosts.
  • the data generated in the detection assay is more reliable and useful when sensitivity limits are established, providing the reviewer of such data an improved opportunity to draw conclusions necessary for making informed decisions.
  • Levine, M.M., et al., Journal Infectious Diseases 148:699-709 (1983) refer to the recombinant DNA risk assessment studies in humans; the efficacy of poorly mobilizable plasmids in biologic containment.
  • Bacterial strains are used that contain selectable markers which render them sensitive under certain selectable conditions and which also contain plasmids with selectable markers that render them resistant under certain selectable conditions.
  • the bacteria are orally introduced in human subjects. Bacteria from the stools of the subjects are cultured under conditions that select against the introduced bacteria strains but select for bacterial population carrying the introduced plasmids.
  • Bacteria which grow under such conditions are scored as endogenous bacteria which received the resistance selectable plasmid by transconjugatio Stotzky, G. and H. Babich, Recomb. DNA Tech. Bull., 7:163-188 (1984), refer to the fat of genetically-engineered microbes in natural environments. Studies involving the survival o recombinant microbes and DNA are reviewed. The studies relate to determining the presence o recombinant bacteria that contain selectable markers and the presence of DNA cont ⁇ dning selectabl markers.
  • mice refer to the effect o colonizing mice with laboratory and wild type strains of E. coli containing tumor virus genomes Laboratory and wild type strains of E. coli which contained recombinant polyoma virus DNA ar introduced into mice.
  • the mice are evaluated for the development of polyoma infection. Th presence of the recombinant bacteria is determined by plating enteric bacteria derived from th mice selecting for selectable markers present in the recombinant strains.
  • hybridization assays are performed detecting the presence of the polyoma DNA. The sensitivit of the hybridization assay is neither disclosed or discussed.
  • the present invention relates to the use of lysogen comprising a single copy of a gene o interest and a selectable marker to determine the sensitivity of an assay, detect a gene of aus in a heterogenous population of bacteria.
  • the present invention relates to a method o determining the level of sensitivity of an assay that is used to detect bacteria carrying a gene o interest in a heterologous population of bacteria.
  • the method comprises the steps of producing a mixed population of bacteria by mixing a population of lysogens that comprise a gene of interest and a selectable marker with a population of non-lysogens having neither the gene of interest nor the selectable marker.
  • the number of lysogens and the number of non-lysogens in the mixed population is determined and the mixed population is plated under conditions which select for presence of the selectable marker present.
  • the plated colonies are additionally checked for the presence of the gene of interest.
  • a lysogen is constructed that contains a gene of interest and a selectable marker.
  • the lysogen can be generated from a strain of bacteria capable of surviving in the gut of an animal which is to be the subject of in vivo studies.
  • the choice of strains is not restricted if the sensitivity assay is to be performed solely as an in vitro assay.
  • the term "lysogen" refers to a bacterial strain in which a foreign piece of
  • Lysogens are generated by infecting a bacterial cell with a recombinant bacteriophage (phage) that contains a gene of interest and a selectable marker.
  • phage recombinant bacteriophage
  • the phage DNA integrates into the bacteria's chromosome.
  • gene of interest refers to a gene which is to be tracked or detected. Often, a gene of interest encodes a desired protein and the gene of interest is inserted in a production host in order to produce the protein by recombinant means.
  • assays are performed to determine the capability of a gene of interest's to move from one host strain to another, the latter strain being one capable of surviving in an animal host.
  • assays are performed to determine whether a bacterial strain which carries a gene of interest is capable of surviving in a host animal.
  • a gene of interest usually within the production host, is introduced into a potential animal host, usually orally.
  • the microbial flora in the animal's fecal matter is then examined for the presence of the gene of interest. If the gene of interest is located, either it has been taken up by an endogenous bacterial strain or the introduced strain is capable of surviving in the host animal's gut.
  • the assay used to detect the gene of interest comprises growing the fecal matter-derived bacteria and employing means to indicate the presence of the gene of interest.
  • Various detection means are available.
  • the most common and straight forward way to detect the presence of a gene of interest is by hybridization using a radiolabelled probe.
  • a probe comprises a radiolabelled copy of the gene of interest or a portion thereof. Production of probes useful in hybridization techniques are well known to those having ordinary skill in the art.
  • the fecal matter-derived bacteria are grown in single colonies using agar media.
  • a single colony is a clonal colony where every cell contains identical genetic material and each cell in the colony is derived from a single parent cell.
  • plaque refers to the techniques for growing single colonies. Plating techniques are well known to those having ordinary skill in the art. Briefly, bacteria is diluted into a range of dilutions and added to petri dishes or plates which contain agar media. At the proper dilution the bacteria are diluted sufficiently such that they are separated from each other at a g at enough distance so that resulting colonies are discrete and all cells in a colony are derived from the single cell. Techniques involving plating on nitrocellulose paper and performing colony lifts onto nitrocellulose paper are also well known to those having ordinary skill in the art.
  • the in vitro hybridization assay described here is developed to address in vitro the impact of plasmid transfer in a heterogenous population of mouse gut E. coli.
  • the present invention relates to the use of a lysogen to determine the sensitivity of an assa which is used to detect a gene of interest in a heterogenous population of bacteria.
  • the presen invention also relates to a method of determining the level of sensitivity of an assay which is use to detect bacteria carrying a gene of interest in a heterologous population of bacteria. Th techniques used to practice the present invention are well known to those having ordinary skill i the art and can be performed using readily available starting materials.
  • a lysogen is constructed which contains a gene of ref and at least one selectable marker. It is, however, preferable that the lysogen contain tw selectable markers, one of which is closely adjacent to the gene of interest.
  • the lysogen i constructed by infecting a bacterial strain with a bacteriophage which does not enter the lyti growth cycle, i.e., the presence of the phage in the host does not result in the production o infective particles. Rather, the phage DNA integrates into the bacterial chromosomal DNA an is referred to as a prophage. It is preferred that the bacteriophage carries both a copy of the gen of interest and a selectable marker.
  • the gene of interest and the selectable marker are adjacent genetic recombination of the lysogen can be determined. Genetic recombination of the phage DN in such lysogens is indicated if the lysogens are detected using antibiotic selection but do no contain the gene of interest as detected by a hybridization assay. If a second selectable marker i provided, it can be present anywhere in the bacteria cell, either on the chromosome or on plasmid within the host cell. The presence of two selectable markers allows for the selection o a small number of slow replicating lysogens when mixed with a larger number of non-lysoge bacteria.
  • a lysogen starter culture and non-lysogen starter culture are grown up.
  • Various known quantities of lysogen starter culture are diluted and mixed with various known quantities of non- lysogen starter culture.
  • the mixtures of cultures referred to herein as the "mixed populations" are plated on nitrocellulose filters over agar plates that contain selection media useful to select for lysogens. If the lysogen contains two selection markers, the agar plates contain media that selects for both markers.
  • the starter cultures are both plated on non-selection agar and colonies are grown up and counted. Determining the concentrating bacteria in culture by plating is routine. Once the concentration is determined, the number of lysogens mixed with non-lysogens when the two are mixed can be determined.
  • the colonies which grow on the selection plates from the plating of the mixed population are lysogen. The colonies are counted. Finally, nitrocellulose lifts of the mixed population plates are made and tested by hybridization assay using a probe to the gene of interest.
  • the data generated from the colony counts and hybridization assay can be used to determine the level of sensitivity of the detection assays. That is, comparing the expected results which can be calculated with the actual results which are observed, the level at which these two sets of data agree is the level of sensitivity of the detection assay. Beyond the level of sensitivity, observed results cannot be considered reliable. However, within the level of sensitivity, results can be accepted as accurate and true.
  • the experimentation designed to address these environmental concerns necessitated the use of a bacterial strain capable of colonizing the murine gastrointestinal tract and modified to carry the BSt cDNA in a single chromosomal copy.
  • the use of a lysogenized strain which carried both the antibiotic resistance of the non-lysogenized parent strain as well as an alternative antibiotic resistance to the parent strain conferred by the prophage has the added benefit of selecting for the alternative antibiotic marker and/or the BSt insert.
  • the added advantage of the double antibiotic resistance of the lysogen is the ability to select for the lysogen out of a background population of the colonized parent strain.
  • the prophage antibiotic resistance marker could be used to determine if the BSt insert have segregated from the host chromosome. Thus, a primary screen for antibiotic resistance could be established followed by the secondary hybridization screen.
  • the in vitro experimentation according to the present invention is designed to assess th sensitivity of the hybridization assay and whether or not this level of sensitivity could b determined from lysogens grown on both non-selective and selective media.
  • the embodiment of the present invention described herein consists of three components; 1) use of an existing bacterial phage lambda to create a phage containing a copy of the bovine somatotropin (BSt) gene; 2) use of the bovine somatotropin gene carrying phage to infect wildtype E. coli cells creating BSt phage lysogens; and, 3) use of the BSt lysogens in an existing murine animal model to demonstrate colonization of the GI tract by the cells and subsequent use of these strains in this model as a component of an environmental assessment study required by the Federal Food and Drug Administration for the approval of bovine somatotropin.
  • BSt bovine somatotropin
  • Reagents used in the preparation of solutions are of reagent grade or better.
  • Ampicillin and streptomycin as well as Kodak XAR-5 film for autoradiography is purchased from Sigma Chemicals, St. Louis, Mo.
  • MacConkey agar is purchased from Becton Dickinson, Cockeysville, MD, and prepared as per manufacturers recommendations.
  • Falcon 100 X 15 mm polystyrene petri dishes are purchased from Becton Dickinson Labware, Lincoln Park, NJ.
  • Nitrocellulose filter membranes (S&S #20460 BA85; .45um pore size; 82.5mm diameter) for DNA blotting is obtained from Schleicher and Schuell, Keene, NH.
  • Random primer labelling reagents are obtained from Stratagene, La Jolla, CA. 32 P-dCTP is obtained from Amersham, Chicago, IL. Whatman 3MM chromatography'paper is purchased from VWR Scientific, Piscataway, NJ. Analysis of variance using a 2 X 2 X 2 factorial design is conducted using SAS software, copyright 1984,1986; SAS Institute Inc., Cary, N.C. MacConkey agar plates are prepared as per manufacturers instructions. In some cases the molten media is supplemented with either streptomycin (100 /ig/ml) or streptomycin (100 ⁇ g/ml) and ampicillin (100 ⁇ g/ml). The entire contents of all references cited below are hereby incorporated by reference. Construction of lysogens is described below. First, a phage carrying the BST cDNA is constructed. It is then used to generate a bacteria lysogen.
  • ⁇ phage BDC531 is obtained from Dr. Donald Court, Frederick Cancer Institute, Frederick MD. This phage is employed in a homologous recombination experiment with cells transformed with the BSt plasmid, pLEBGH-Z.
  • the BSt cDNA is described in Tomich, C-S. C, et al., Plasmid 20:167-170 (1988). From this experiment, a new population of phage carrying the bovine somatotropin gene are created. Purified BSt phage are subsequently modified in a fashion which prevented their entering a lytic growth cycle when present as prophage in the bacterial chromosome.
  • Phage generated in this series of experiments are assigned the laboratory designations XBST21 and XBST434.
  • Infective ⁇ phages BST21 and BST434 are used to generate lysogens in E. coli cells isolated from murine gastrointestinal tract.
  • the E. coli isolates employed are designated and are . made resistant to the antibiotic streptomycin by selection of spontaneous mutants on streptomycin plates.
  • three E. coli isolates are infected in separate experiments with XBST21 and XBST434 phage to generate a total of six lysogenic strains capable of colonizing murine gastrointestinal tract.
  • Cells lysogenized with XBST434 are preferred for in vivo applications because of their genotype which prevents prophage from entering a lytic growth cycle.
  • the ability of the hybridization assay to detect XBST434 lysogens designated strains UC12692 and UC12693 in a background of non-lysogens designated strains UC12699 and UC12700 is assessed by plating mixed populations of lysogens and non-lysogens on nitrocellulose filters over on agar plates containing selective media and subsequently probing filter lifts prepared from these plates with radiolabelled BSt cDNA.
  • the inocula for these mixtures are prepared by inoculating beads from frozen stocks into brain heart in&sion broth (BHIB) containing either 100 ⁇ g streptomycin/ml (strains UC12699 and UC12700) or into BHIB containing 100 ⁇ g streptomycin ml and 10 ⁇ g ampicillin/ml (strains UC12692 and UC12693). These cultures are incubated at 36° C without agitation for 16-18 hours before dilution and mixing. The exact inoculum size is determined for each culture by viable plate counts on plain MacConkey agar medium plates.
  • Non-lysogenized strains UC12699 and UC12700 are diluted to 2.9 and 3.6 X 10 8 cfu/ml, respectively, and mixed with a population of the corresponding XBST434 lysogen (UC12692 or
  • the plates for the hybridization assays are prepared by inoculating 0.1 ml of the mixtures directly onto 82.5 mm nitrocellulose membrane filters which have been overlayed on MacConkey agar supplemented with both ampicillin (100 ⁇ g/ ml) and streptomycin (100 ⁇ g/ml). Ten plates per dilution are prepared and these are incubated 16-18 hours at 36° C. Original inoculum densities for each culture is determined by dilution and plating onto plain MacConkey agar. Colony counts are recorded directly from these same plates which are also used for the hybridization assay.
  • the bacterial lawn grew on the surface of th nitrocellulose filter itself thereby eliminating much of the smearing associated with performing a filter lift from cells grown on an agar surface.
  • Ten plates for each lysogen dilution are prepared in this fashion on MacConkey ampicillin/streptomycin agar. These plates are designated Master Plates from which duplicated filters are prepared and probed with the radiolabelled BSt insert.
  • Duplicated filters are prepared from the Master Plates and grown at 37° C for 2 hours in the presence of streptomycin and ampicillin.
  • viable colony counts are obtained by direct visual inspection of 0.5 - 1.0 mm diameter colonies. After obtaining the colony counts, one nitrocellulose lift is conducted on each of the ampicillin/streptomycin plates containing the nitrocellulose membranes and grown at 37° C for 2 hours on ampicillin/streptomycin MacConkey plates. The nitrocellulose membranes on which the bacterial mixes have been grown are used as one of the duplicate membranes for the hybridization assay so a direct comparison between colony counts and hybridization signal could be made.
  • Membranes containing the bacterial colony lifts are lysed, neutralized, and washed according to the method of Sambrook, J., et al., "Molecular Cloning: A Laboratory Manual, Coldspring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) by successively placing filters for 5 minutes onto 3MM paper saturated with: 1) 0.5N sodium hydroxide/1.5M sodium chloride; 2) 0.5M Tris-hydrogen chloride ⁇ H 7.4 at 25°C)/1.5 M sodium chloride; 3) 2X SSC.
  • the filters are dried at room temperature for 1 hour, sandwiched between sheets of 3MM paper, baked for 1.5 hours at 80° C/25 inches of vacuum, and stored in the dark at room temperature until screened for the presence of BSt cDNA.
  • Screening of all colonies for BSt cDNA employed the Clal/BamHI restriction fragment of plasmid pURA-4 (pUC1195) containing the BSt coding sequence as described in European Patent Publication 0 418 219. This fragment is radiolabelled to a specific activity of 1-2 X 10 9 cpms/ ⁇ g using the random primer method of Feinberg, A. P and Vogelstein, B., Anal.
  • Prehybridization volume is set at 0.2 mis for each square centimeter of filter surface or 10.6 mis per 82.5 mm filter.
  • the 32 P random primer labelled Clal/BamHI restriction fragment is heat denatured at 100°C for 5 minutes followed by rapid chilling in ice water.
  • the heat denatured probe is added to the prehybridization solution covering the filters at a concentration of 1 X 10 6 cpms/ml and rotated at 68°C for 20 hours.
  • the filters are washed successively in 2 liters (4 X 500 ml washes for 5 minutes/wash) of 2X SSC/0.1% SDS at 22°C, 500 mis of IX SSC/0.1% SDS for 1 hour at 68° C, and 500mls of 0.2X SSC/0.1% SDS for 1 hour at 68° C.
  • the experiment disclosed here further defined the sensitivity of the hybridization assay as being able to detect one XBST434 lysogen within a non-lysogen background population of approximately 4 X 10 8 cfu. This is importan because it extends the operational range of this type of assay to a level well below that require for in vivo experiments designed to address the question of potential plasmid transfer in heterogenous population of E. coli.

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Abstract

On utilise du lysogène contenant une seule copie du gène recherché et un marqueur sélectionnable pour déterminer la sensibilité d'une technique utilisée pour détecter un gène recherché dans une population hétérogène de bactéries. Cette invention concerne également un procédé permettant de déterminer le niveau de sensibilité d'une technique utilisée pour détecter dans une population hétérologue de bactéries, des bactéries porteuses d'un gène recherché.
PCT/US1992/006535 1991-08-29 1992-08-11 Procede permettant de determiner la sensibilite d'une technique de detection d'adn WO1993005171A1 (fr)

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US75119791A 1991-08-29 1991-08-29
US751,197 1991-08-29

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1986005209A1 (fr) * 1985-03-05 1986-09-12 James William Blackburn Procede de controle et de commande de populations microbiennes

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1986005209A1 (fr) * 1985-03-05 1986-09-12 James William Blackburn Procede de controle et de commande de populations microbiennes

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
PLASMID vol. 20, no. 2, September 1988, NEW YORK, US pages 167 - 170 C-S. C. TOMICH ET AL. 'Use of lacZ expression to monitor transcription' cited in the application *
RECOMBINANT DNA TECHNOLOGY BULLETIN vol. 7, December 1984, BETHESDA MD, US pages 163 - 188 G. STOTZKY ET AL. 'Fate of genetically-engineered microbes in natural environments' cited in the application *
RECOMBINANT DNA TECHNOLOGY BULLETIN vol. 8, June 1985, BETHESDA MD, US pages 47 - 51 C. SMITH ET AL. 'The effect of colonizing mice with laboratory and wild type strains of E. coli containing virus genomes' cited in the application *

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