Method For Genetic Selection Of High-Plasmid Producing E. Coli Clones
The present invention relates to methods of selecting for highly productive clones of E. coli for the production of plasmid DNA comprising measuring the frequency of IS1 transposon insertional mutagenesis within either the plasmid or genomic DNA of transformed clonal subtypes. An increase in IS1 insertional mutagenesis is correlated with clonal subtypes likely to exhibit a low specific productivity. The PCR-based, genetic selection assays disclosed herein are amenable to high throughput analysis, reducing the time to identify highly productive clones capable of cultivating large quantities of plasmid DNA on an industrial scale.
1 . A method for selecting a highly productive clonal subtype of a strain of E. coli harboring a plasmid DNA comprising:
(a) comparing IS1 transposition activity in at least two clonal subtypes of the same strain harboring the same plasmid DNA, wherein the clonal subtype that displays a comparatively lower transposition activity represents a potential highly productive clonal subtype; and,
(b) testing productivity of said potential highly productive clonal subtype;
wherein a highly productive clonal subtype exhibits a high plasmid copy number per cell.
2 . A method of claim 1 , wherein IS1 transposition activity is determined by measuring IS1 transposon copy number in isolated plasmid DNA samples from said clonal subtypes, wherein a comparatively lower IS1 transposon copy number indicates a comparatively lower IS1 transposition activity.
3 . A method of claim 1 , wherein IS1 transposition activity is determined by measuring the presence or absence of an IS1 transposon sequence in a predetermined IS1 insertion region within genomic DNA of said clonal subtypes, wherein the absence of an IS1 insertion sequence indicates a comparatively lower IS1 transposition activity.
4 . A method for selecting a highly productive clonal subtype of a strain of E. coli harboring a plasmid DNA comprising:
(a) isolating plasmid DNA from at least two clonal subtypes of the same strain harboring the same plasmid DNA;
(b) measuring IS1 transposon copy number in said isolated plasmid DNA samples, wherein the clonal subtype that displays a comparatively lower IS1 transposon copy number represents a potential highly productive clonal subtype; and
(c) testing productivity of said potential highly productive clonal subtype; wherein a highly productive clonal subtype exhibits a high plasmid copy number per cell.
5 . A method of claim 4 , wherein the IS1 transposon copy number is measured using a quantitative PCR assay.
6 . A method of claim 5 , wherein the quantitative PCR assay measures the relative quantity of IS1 based on plasmid copy number by amplifying both a first nucleotide sequence of the plasmid DNA located within the IS1 nucleotide sequence and a second nucleotide sequence of the plasmid DNA determined to be free of IS1 insertions, generating an IS1/plasmid copy ratio which represents the IS1 transposon copy number.
7 . A method of claim 6 , wherein the IS1/plasmid copy ratio is corrected by subtracting the predicted quantity of IS1 transposon copies contributed from residual genomic DNA present in the plasmid DNA sample.
8 . A method of claim 7 , wherein the predicted quantity of IS1 transposon copies contributed from residual genomic DNA present in the plasmid DNA sample is measured using a second quantitative PCR assay.
9 . A method of claim 8 , wherein the second quantitative PCR assay measures the relative quantity of 23s rDNA based on plasmid copy number by amplifying both a nucleotide sequence of the residual genomic DNA located within the 23s rDNA nucleotide sequence and the second nucleotide sequence of the plasmid DNA determined to be free of IS1 insertions used to generate the IS1/plasmid copy ratio, generating a 23s rDNA/plasmid copy ratio that is subtracted from the IS1/plasmid copy ratio to provide a corrected IS1/plasmid copy ratio.
10 . A method of claim 6 , wherein the first and second nucleotide sequences of the plasmid DNA are individually amplified in the presence of a nucleic acid polymerase and a set of oligonucleotides, wherein the set of oligonucleotides used to amplify the first nucleotide sequence consists of:
(i) a forward PCR primer that hybridizes to a first location of the IS1 nucleotide sequence;
(ii) a reverse PCR primer that hybridizes to a second location of the IS1 nucleotide sequence downstream of the first location; and,
(iii) a fluorescent probe labeled with a quencher molecule and a fluorophore which emits energy at a unique emission maxima, said probe hybridizes to a location within the IS1 nucleotide sequence between the first and second locations;
and the set of oligonucleotides used to amplify the second nucleotide sequence consists of:
(i) a forward PCR primer that hybridizes to a first location of the second nucleotide sequence;
(ii) a reverse PCR primer that hybridizes to a second location of the second nucleotide sequence downstream of the first location; and
(iii) a fluorescent probe labeled with a quencher molecule and a fluorophore which emits energy at a unique emission maxima, said probe hybridizes to a location within the second nucleotide sequence between the first and second locations;
wherein said nucleic acid polymerase digests the fluorescent probes during amplification to dissociate said fluorophores from said quencher molecules, and a change of fluorescence upon dissociation of the fluorophore and quencher molecules is detected, the change of fluorescence corresponding to the occurrence of amplification of the first and/or second nucleotide sequences.
11 . (canceled)
12 . A method of claim 10 , wherein the set of oligonucleotides used to amplify the first nucleotide sequence consists of forward and reverse PCR primers IS1-Q-F (SEQ ID NO:6) and IS1-Q-R (SEQ ID NO:7), respectively, and fluorescent probe IS1-Q-P2 (SEQ ID NO:8); and the set of oligonucleotides used to amplify the second nucleotide sequence consists of forward and reverse PCR primers CMV-Q-F (SEQ ID NO:3) and CMV-Q-R (SEQ ID NO:4), respectively, and fluorescent probe CMV-Q-P2 (SEQ ID NO:5).
13 . (canceled)
14 . A method of claim 9 , wherein the nucleotide sequence located within the 23s rDNA sequence and the second nucleotide sequence are individually amplified in the presence of a nucleic acid polymerase and a set of oligonucleotides, wherein the set of oligonucleotides used to amplify the first nucleotide sequence consists of:
(i) a forward PCR primer that hybridizes to a first location of the 23s rDNA sequence;
(ii) a reverse PCR primer that hybridizes to a second location of the 23s rDNA sequence downstream of the first location; and,
(iii) a fluorescent probe labeled with a quencher molecule and a fluorophore which emits energy at a unique emission maxima; said probe hybridizes to a location within the 23s rDNA sequence between the first and second locations;
and the set of oligonucleotides used to amplify the second nucleotide sequence consists of:
(iv) a forward PCR primer that hybridizes to a first location of the second nucleotide sequence;
(v) a reverse PCR primer that hybridizes to a second location of the second nucleotide sequence downstream of the first location; and
(vi) a fluorescent probe labeled with a quencher molecule and a fluorophore which emits energy at a unique emission maxima, said probe hybridizes to a location within the second nucleotide sequence between the first and second locations;
wherein said nucleic acid polymerase digests the fluorescent probes during amplification to dissociate said fluorophores from said quencher molecules, and a change of fluorescence upon dissociation of the fluorophore and quencher molecules is detected, the change of fluorescence corresponding to the occurrence of amplification of the 23s rDNA sequence and/or second nucleotide sequence.
15 . (canceled)
16 . A method of claim 14 , wherein the set of oligonucleotides used to amplify the 23s rDNA nucleotide sequence consists of forward and reverse PCRprimers 23s-F1D (SEQ ID NO:11) and 23s-RID (SEQ ID NO:12), respectively, and fluorescent probe 23s-Pfam (SEQ ID NO: 13); and the set of oligonucleotides used to amplify the second nucleotide sequence consists of forward and reverse PCR primers CMV-Q-F (SEQ ID NO:3) and CMV-Q-R (SEQ ID NO:4), respectively, and fluorescent probe CMV-Q-P2 (SEQ ID NO:5).
17 . (canceled)
18 . A method for selecting a highly productive clonal subtype of a strain of E. coli harboring a plasmid DNA comprising:
(a) detecting the presence or absence of an IS1 transposon sequence within a predetermined IS1 insertion region of the genomic DNA of said clonal subtype, wherein a clonal subtype lacking an IS1 transposon sequence within said IS1 insertion region represents a potential highly productive clonal subtype; and,
(b) testing productivity of said potential highly productive clonal subtype; wherein a highly productive clonal subtype exhibits a high plasmid copy number per cell.
19 . A method of claim 18 , wherein said IS1 insertion region spans less than about 20 contiguous nucleotides of the genomic DNA.
20 . A method of claim 19 , wherein a quantitative PCR assay is used to detect the presence or absence of an IS1 transposon sequence within said IS1 insertion region of the genomic DNA.
21 . A method of claim 20 , wherein the quantitative PCR assay amplifies a portion of the genomic DNA that contains the IS1 insertion region in the presence of a nucleic acid polymerase and a set of oligonucleotides consisting of:
(i) a fluorescent probe labeled with a quencher molecule and a fluorophore which emits energy at a unique emission maxima, wherein said probe hybridizes to a location within the genomic DNA that spans the IS1 insertion region only when said genomic DNA lacks an IS1 transposon sequence within said IS1 insertion region;
(ii) a forward PCR primer that hybridizes to a location of the genomic DNA upstream of the fluorescent probe; and,
(iii) a reverse PCR primer that hybridizes to a location of the genomic DNA downstream of the fluorescent probe;
wherein said nucleic acid polymerase digests the fluorescent probe during amplification to dissociate said fluorophore from said quencher molecule, and a change of fluorescence upon dissociation of the fluorophore and the quencher molecule is detected, the change of fluorescence corresponding to amplification of the genomic DNA and the absence of an IS1 transposon sequence within the IS1 insertion region.
22 . A method of claim 21 , wherein said quantitative PCR assay is performed on a whole cell lysate.
23 . A method of claim 18 , wherein said IS1 insertion region spans greater than about 20 contiguous nucleotides of the genomic DNA.
24 . A method of claim 23 , wherein a PCR assay is used to detect the presence or absence of an IS1 transposon sequence within said IS1 insertion region of the genomic DNA.
25 . A method of claim 24 , wherein the PCR assay amplifies a portion of the genomic DNA in the presence of a nucleic acid polymerase and a set of oligonucleotides consisting of:
(i) a first PCR primer that hybridizes to a location of the genomic DNA outside of the IS1 insertion region; and,
(ii) a second PCR primer that hybridizes to a location within an IS1 transposon sequence inserted within the IS1 insertion region;
wherein the presence of an IS1 transposon sequence within the IS1 insertion region results in exponential amplification of said portion of the genomic DNA due to the hybridization of both PCR primers, and the absence of an IS1 transposon sequence within the IS1 insertion region results in linear amplification of only a single strand of said portion of the genomic DNA due to hybridization of only the first PCR primer.
26 . A method of claim 25 , wherein amplification of said portion of the genomic DNA is visually detected by identifying amplified nucleic acid fragments of approximate target size.
27 . A method of claim 25 , wherein amplification of said portion of the genomic DNA is fluorescently detected in real-time by adding a nucleic acid stain that binds double-stranded DNA.