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Escherichia coli MW005: lambda Red-mediated recombineering and copy-number induction of oriV-equipped constructs in a single host.

Westenberg M, Bamps S, Soedling H, Hope IA, Dolphin CT - BMC Biotechnol. (2010)

Bottom Line: BAC and fosmid clones are highly suitable for modification by recombineering but, because they are present at low (1-2) copies per cell, the DNA is difficult to isolate in high yield and purity.Previously, we used EL350 and EPI300 sequentially to recombineer oriV-equipped fosmid genomic clones and, subsequently, to induce copy-number of the resulting recombinant clone.The PBAD-driven copy of cre in EL350 was replaced seamlessly with a copy of trfA, PCR-amplified from EPI300 chromosomal DNA, to generate MW005.

View Article: PubMed Central - HTML - PubMed

Affiliation: Pharmaceutical Science Division, King's College London, 150 Stamford Street, London, SE1 9NH, UK.

ABSTRACT

Background: Escherichia coli strain EL350 contains chromosomally integrated phage lambda Red recombinase genes enabling this strain to be used for modifying the sequence of resident clones via recombineering. BAC and fosmid clones are highly suitable for modification by recombineering but, because they are present at low (1-2) copies per cell, the DNA is difficult to isolate in high yield and purity. To overcome this limitation vectors, e.g. pCC1FOS, have been constructed that contain the additional replication origin, oriV, which permits copy-number to be induced transiently when propagated in a suitable host strain, e.g. EPI300, that supplies the cognate trans-replication protein TrfA. Previously, we used EL350 and EPI300 sequentially to recombineer oriV-equipped fosmid genomic clones and, subsequently, to induce copy-number of the resulting recombinant clone. To eliminate these intervening DNA isolation and transformation steps we retrofitted EL350 with a PBAD-driven trfA gene generating strain MW005 that supports, independently, both recombineering and copy-number induction.

Results: The PBAD-driven copy of cre in EL350 was replaced seamlessly with a copy of trfA, PCR-amplified from EPI300 chromosomal DNA, to generate MW005. This new strain has been used to both generate, via recombineering, a number of reporter gene fusions directly from pCC1FOS-based Caenorhabditis elegans genomic clones and to transiently induce copy-number of fosmid and BAC clones prior to DNA preparation.

Conclusions: By retrofitting EL350, an established 'recombineering' E. coli strain, with a tightly regulated copy of trfA we have produced a new strain, MW005, which combines recombineering capacity with the useful ability to transiently induce copy-number of oriV-equipped clones. By coupling these two steps in a single strain, use of MW005 will enable the more rapid recombineering-mediated production of recombinant clones in the yield and quality necessary for many downstream purposes.

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Copy-number induction in MW005. Panel A. Aliquots (5 μl) of fUL#SB28 (gel i), WRM0636aA04 (gel ii) or WRM067aC01 (gel iii) pCC1FOS-based DNA, isolated from equivalent numbers of cells from either non-induced (NI) or copy-number-induced (I) cultures of EPI300 or MW005 and incubated with either BamHI (gel i) or NcoI (gels ii & iii), were electrophoresed through a 0.7% (w/v) agarose gel either undiluted (lanes 1, 8) or after 2- (lanes 2, 9), 4- (lanes 3, 10), 8- (lanes 4, 11), 16- (lanes 5, 12), 32- (lanes 6, 13) or 64-fold (lanes 7, 14) dilution. Panel B. Aliquots (5 μl) of A02_CBP0333 (gel i), A10_CBP1191 (gel ii) or H12_CBP0642 (gel iii) pCC1BAC-based DNA, isolated from equivalent numbers of cells from either non-induced (NI) or copy-number-induced (I) cultures of EPI300 or MW005 and incubated with NcoI, were electrophoresed through a 0.7% (w/v) agarose gel either undiluted (lanes 1, 7) or after 2- (lanes 2, 8), 4- (lanes 3, 9), 8- (lanes 4, 10), 16- (lanes 5, 11) or 32-fold (lanes 6, 12) dilution. M = DNA ladder (kb).
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Figure 2: Copy-number induction in MW005. Panel A. Aliquots (5 μl) of fUL#SB28 (gel i), WRM0636aA04 (gel ii) or WRM067aC01 (gel iii) pCC1FOS-based DNA, isolated from equivalent numbers of cells from either non-induced (NI) or copy-number-induced (I) cultures of EPI300 or MW005 and incubated with either BamHI (gel i) or NcoI (gels ii & iii), were electrophoresed through a 0.7% (w/v) agarose gel either undiluted (lanes 1, 8) or after 2- (lanes 2, 9), 4- (lanes 3, 10), 8- (lanes 4, 11), 16- (lanes 5, 12), 32- (lanes 6, 13) or 64-fold (lanes 7, 14) dilution. Panel B. Aliquots (5 μl) of A02_CBP0333 (gel i), A10_CBP1191 (gel ii) or H12_CBP0642 (gel iii) pCC1BAC-based DNA, isolated from equivalent numbers of cells from either non-induced (NI) or copy-number-induced (I) cultures of EPI300 or MW005 and incubated with NcoI, were electrophoresed through a 0.7% (w/v) agarose gel either undiluted (lanes 1, 7) or after 2- (lanes 2, 8), 4- (lanes 3, 9), 8- (lanes 4, 10), 16- (lanes 5, 11) or 32-fold (lanes 6, 12) dilution. M = DNA ladder (kb).

Mentions: To investigate whether MW005 would support copy-number induction of oriV-equipped clones we compared copy-number induction in EPI300 and MW005 for three pCC1FOS-based clones, the final recombineered C. elegans gene fusion reporter fUL#SB28 (see below) plus two native genomic clones, and three pCC1BAC-based genomic clones from a library constructed with Lates calcarifer (Barramundi) genomic DNA (kind gift of G.H. Yue). DNAs, isolated from equal numbers of cells from EPI300 or MW005 cultures that were either non-induced for copy-number or had received L-arabinose to drive TrfA expression and thus induce copy-number, were restricted and electrophoresed. Copy-numbers of pCC1FOS- and pCC1BAC-based clones were induced in both EPI300 and MW005 by an approximately equal extent (Fig. 2). For each clone, careful visual comparison between the ethidium bromide-stained restriction fragments of DNA isolated from the control culture with those of a 2-fold serial dilution of the equivalent fragments of DNA isolated from the induced culture enabled fold-induction to be estimated for both strains. Such examination indicates that the copy-numbers of all three pCC1FOS-based clones were induced from 50-60-fold in both the commercial EPI300 strain and MW005, e.g. in Fig. 2A, gels i, ii and iii, compare lane 7, containing the restriction digest, diluted 1/64, from induced EPI300, with lane 1, containing the undiluted restriction digest from non-induced EPI300, and lanes 14 and 8 containing, respectively, the equivalent restriction digest dilutions for induced and non-induced MW005. Although, for the three pCC1BAC-based clones, there was some slight variation in copy-number induction between clones and strains, visual examination of Fig. 2B indicates that, for each BAC clone, copy-number was increased approximately 15-20-fold, e.g. compare lane 5, containing the 1/16-diluted restriction digest from induced EPI300, with lane 1, containing the undiluted restriction digest from non-induced EPI300, and lanes 11 and 7 containing the equivalent dilutions for induced and non-induced MW005, respectively.


Escherichia coli MW005: lambda Red-mediated recombineering and copy-number induction of oriV-equipped constructs in a single host.

Westenberg M, Bamps S, Soedling H, Hope IA, Dolphin CT - BMC Biotechnol. (2010)

Copy-number induction in MW005. Panel A. Aliquots (5 μl) of fUL#SB28 (gel i), WRM0636aA04 (gel ii) or WRM067aC01 (gel iii) pCC1FOS-based DNA, isolated from equivalent numbers of cells from either non-induced (NI) or copy-number-induced (I) cultures of EPI300 or MW005 and incubated with either BamHI (gel i) or NcoI (gels ii & iii), were electrophoresed through a 0.7% (w/v) agarose gel either undiluted (lanes 1, 8) or after 2- (lanes 2, 9), 4- (lanes 3, 10), 8- (lanes 4, 11), 16- (lanes 5, 12), 32- (lanes 6, 13) or 64-fold (lanes 7, 14) dilution. Panel B. Aliquots (5 μl) of A02_CBP0333 (gel i), A10_CBP1191 (gel ii) or H12_CBP0642 (gel iii) pCC1BAC-based DNA, isolated from equivalent numbers of cells from either non-induced (NI) or copy-number-induced (I) cultures of EPI300 or MW005 and incubated with NcoI, were electrophoresed through a 0.7% (w/v) agarose gel either undiluted (lanes 1, 7) or after 2- (lanes 2, 8), 4- (lanes 3, 9), 8- (lanes 4, 10), 16- (lanes 5, 11) or 32-fold (lanes 6, 12) dilution. M = DNA ladder (kb).
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Related In: Results  -  Collection

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Figure 2: Copy-number induction in MW005. Panel A. Aliquots (5 μl) of fUL#SB28 (gel i), WRM0636aA04 (gel ii) or WRM067aC01 (gel iii) pCC1FOS-based DNA, isolated from equivalent numbers of cells from either non-induced (NI) or copy-number-induced (I) cultures of EPI300 or MW005 and incubated with either BamHI (gel i) or NcoI (gels ii & iii), were electrophoresed through a 0.7% (w/v) agarose gel either undiluted (lanes 1, 8) or after 2- (lanes 2, 9), 4- (lanes 3, 10), 8- (lanes 4, 11), 16- (lanes 5, 12), 32- (lanes 6, 13) or 64-fold (lanes 7, 14) dilution. Panel B. Aliquots (5 μl) of A02_CBP0333 (gel i), A10_CBP1191 (gel ii) or H12_CBP0642 (gel iii) pCC1BAC-based DNA, isolated from equivalent numbers of cells from either non-induced (NI) or copy-number-induced (I) cultures of EPI300 or MW005 and incubated with NcoI, were electrophoresed through a 0.7% (w/v) agarose gel either undiluted (lanes 1, 7) or after 2- (lanes 2, 8), 4- (lanes 3, 9), 8- (lanes 4, 10), 16- (lanes 5, 11) or 32-fold (lanes 6, 12) dilution. M = DNA ladder (kb).
Mentions: To investigate whether MW005 would support copy-number induction of oriV-equipped clones we compared copy-number induction in EPI300 and MW005 for three pCC1FOS-based clones, the final recombineered C. elegans gene fusion reporter fUL#SB28 (see below) plus two native genomic clones, and three pCC1BAC-based genomic clones from a library constructed with Lates calcarifer (Barramundi) genomic DNA (kind gift of G.H. Yue). DNAs, isolated from equal numbers of cells from EPI300 or MW005 cultures that were either non-induced for copy-number or had received L-arabinose to drive TrfA expression and thus induce copy-number, were restricted and electrophoresed. Copy-numbers of pCC1FOS- and pCC1BAC-based clones were induced in both EPI300 and MW005 by an approximately equal extent (Fig. 2). For each clone, careful visual comparison between the ethidium bromide-stained restriction fragments of DNA isolated from the control culture with those of a 2-fold serial dilution of the equivalent fragments of DNA isolated from the induced culture enabled fold-induction to be estimated for both strains. Such examination indicates that the copy-numbers of all three pCC1FOS-based clones were induced from 50-60-fold in both the commercial EPI300 strain and MW005, e.g. in Fig. 2A, gels i, ii and iii, compare lane 7, containing the restriction digest, diluted 1/64, from induced EPI300, with lane 1, containing the undiluted restriction digest from non-induced EPI300, and lanes 14 and 8 containing, respectively, the equivalent restriction digest dilutions for induced and non-induced MW005. Although, for the three pCC1BAC-based clones, there was some slight variation in copy-number induction between clones and strains, visual examination of Fig. 2B indicates that, for each BAC clone, copy-number was increased approximately 15-20-fold, e.g. compare lane 5, containing the 1/16-diluted restriction digest from induced EPI300, with lane 1, containing the undiluted restriction digest from non-induced EPI300, and lanes 11 and 7 containing the equivalent dilutions for induced and non-induced MW005, respectively.

Bottom Line: BAC and fosmid clones are highly suitable for modification by recombineering but, because they are present at low (1-2) copies per cell, the DNA is difficult to isolate in high yield and purity.Previously, we used EL350 and EPI300 sequentially to recombineer oriV-equipped fosmid genomic clones and, subsequently, to induce copy-number of the resulting recombinant clone.The PBAD-driven copy of cre in EL350 was replaced seamlessly with a copy of trfA, PCR-amplified from EPI300 chromosomal DNA, to generate MW005.

View Article: PubMed Central - HTML - PubMed

Affiliation: Pharmaceutical Science Division, King's College London, 150 Stamford Street, London, SE1 9NH, UK.

ABSTRACT

Background: Escherichia coli strain EL350 contains chromosomally integrated phage lambda Red recombinase genes enabling this strain to be used for modifying the sequence of resident clones via recombineering. BAC and fosmid clones are highly suitable for modification by recombineering but, because they are present at low (1-2) copies per cell, the DNA is difficult to isolate in high yield and purity. To overcome this limitation vectors, e.g. pCC1FOS, have been constructed that contain the additional replication origin, oriV, which permits copy-number to be induced transiently when propagated in a suitable host strain, e.g. EPI300, that supplies the cognate trans-replication protein TrfA. Previously, we used EL350 and EPI300 sequentially to recombineer oriV-equipped fosmid genomic clones and, subsequently, to induce copy-number of the resulting recombinant clone. To eliminate these intervening DNA isolation and transformation steps we retrofitted EL350 with a PBAD-driven trfA gene generating strain MW005 that supports, independently, both recombineering and copy-number induction.

Results: The PBAD-driven copy of cre in EL350 was replaced seamlessly with a copy of trfA, PCR-amplified from EPI300 chromosomal DNA, to generate MW005. This new strain has been used to both generate, via recombineering, a number of reporter gene fusions directly from pCC1FOS-based Caenorhabditis elegans genomic clones and to transiently induce copy-number of fosmid and BAC clones prior to DNA preparation.

Conclusions: By retrofitting EL350, an established 'recombineering' E. coli strain, with a tightly regulated copy of trfA we have produced a new strain, MW005, which combines recombineering capacity with the useful ability to transiently induce copy-number of oriV-equipped clones. By coupling these two steps in a single strain, use of MW005 will enable the more rapid recombineering-mediated production of recombinant clones in the yield and quality necessary for many downstream purposes.

Show MeSH
Related in: MedlinePlus