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An assay to monitor the activity of DNA transposition complexes yields a general quality control measure for transpositional recombination reactions.

Pulkkinen E, Haapa-Paananen S, Savilahti H - Mob Genet Elements (2014)

Bottom Line: Transposon-based technologies have many applications in molecular biology and can be used for gene delivery into prokaryotic and eukaryotic cells.If transposition targets ccdB, this gene becomes inactivated, enabling plasmid-receiving Escherichia coli cells to survive and to be scored as colonies on selection plates.The colony formation capacity was linearly correlated with the competence status of the E.coli cells, enabling normalization of experimental data obtained with different batches of recipient cells.

View Article: PubMed Central - PubMed

Affiliation: Division of Genetics and Physiology; Department of Biology; University of Turku ; Turku, Finland.

ABSTRACT

Transposon-based technologies have many applications in molecular biology and can be used for gene delivery into prokaryotic and eukaryotic cells. Common transpositional activity measurement assays suitable for many types of transposons would be beneficial, as diverse transposon systems could be compared for their performance attributes. Therefore, we developed a general-purpose assay to enable and standardize the activity measurement for DNA transposition complexes (transpososomes), using phage Mu transposition as a test platform. This assay quantifies transpositional recombination efficiency and is based on an in vitro transposition reaction with a target plasmid carrying a lethal ccdB gene. If transposition targets ccdB, this gene becomes inactivated, enabling plasmid-receiving Escherichia coli cells to survive and to be scored as colonies on selection plates. The assay was validated with 3 mini-Mu transposons varying in size and differing in their marker gene constitution. Tests with different amounts of transposon DNA provided a linear response and yielded a 10-fold operational range for the assay. The colony formation capacity was linearly correlated with the competence status of the E.coli cells, enabling normalization of experimental data obtained with different batches of recipient cells. The developed assay can now be used to directly compare transpososome activities with all types of mini-Mu transposons, regardless of their aimed use. Furthermore, the assay should be directly applicable to other transposition-based systems with a functional in vitro reaction, and it provides a dependable quality control measure that previously has been lacking but is highly important for the evaluation of current and emerging transposon-based applications.

No MeSH data available.


Related in: MedlinePlus

Assay design.In vitro transposition reaction with preassembled transposition complexes and pZErO-2 as a target plasmid. Target plasmid contains kanamycin resistance gene and lethal ccdB gene. In vitro transposition reaction products are introduced into E.coli cells by transformation and selected against kanamycin resistance on antibiotic selection plates. Inactivation of lethal ccdB gene by transposon insertion results into plasmid propagation and antibiotic resistance colonies.
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f0001: Assay design.In vitro transposition reaction with preassembled transposition complexes and pZErO-2 as a target plasmid. Target plasmid contains kanamycin resistance gene and lethal ccdB gene. In vitro transposition reaction products are introduced into E.coli cells by transformation and selected against kanamycin resistance on antibiotic selection plates. Inactivation of lethal ccdB gene by transposon insertion results into plasmid propagation and antibiotic resistance colonies.

Mentions: All transposon-based technologies would greatly benefit from a single activity measurement assay to quantify transposition. To meet this demand for quality control, we used phage Mu DNA transposition as a test platform and set up an in vitro transposition assay that utilizes plasmid pZErO-2 as the target for transposition (Fig. 1.). This plasmid contains a kanamycin resistance cassette for selection in E. coli and a lethal ccdB gene for direct selection of insertions.60-62 Expression of CcdB protein in wild type E. coli cells causes cell death by inhibiting DNA gyrase, an essential enzyme that generates negative supercoils in DNA.63 A transposon insertion into ccdB inactivates the gene, allowing the propagation of the plasmid. In this study, preassembled transpososomes are mixed with pZErO-2 target plasmid and incubated to generate transposition reaction products. The products are then introduced into E. coli cells by transformation, and the cells are selected for kanamycin resistance. The colonies scored represent events where ccdB in the target plasmid has been inactivated.Figure 1.


An assay to monitor the activity of DNA transposition complexes yields a general quality control measure for transpositional recombination reactions.

Pulkkinen E, Haapa-Paananen S, Savilahti H - Mob Genet Elements (2014)

Assay design.In vitro transposition reaction with preassembled transposition complexes and pZErO-2 as a target plasmid. Target plasmid contains kanamycin resistance gene and lethal ccdB gene. In vitro transposition reaction products are introduced into E.coli cells by transformation and selected against kanamycin resistance on antibiotic selection plates. Inactivation of lethal ccdB gene by transposon insertion results into plasmid propagation and antibiotic resistance colonies.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4590003&req=5

f0001: Assay design.In vitro transposition reaction with preassembled transposition complexes and pZErO-2 as a target plasmid. Target plasmid contains kanamycin resistance gene and lethal ccdB gene. In vitro transposition reaction products are introduced into E.coli cells by transformation and selected against kanamycin resistance on antibiotic selection plates. Inactivation of lethal ccdB gene by transposon insertion results into plasmid propagation and antibiotic resistance colonies.
Mentions: All transposon-based technologies would greatly benefit from a single activity measurement assay to quantify transposition. To meet this demand for quality control, we used phage Mu DNA transposition as a test platform and set up an in vitro transposition assay that utilizes plasmid pZErO-2 as the target for transposition (Fig. 1.). This plasmid contains a kanamycin resistance cassette for selection in E. coli and a lethal ccdB gene for direct selection of insertions.60-62 Expression of CcdB protein in wild type E. coli cells causes cell death by inhibiting DNA gyrase, an essential enzyme that generates negative supercoils in DNA.63 A transposon insertion into ccdB inactivates the gene, allowing the propagation of the plasmid. In this study, preassembled transpososomes are mixed with pZErO-2 target plasmid and incubated to generate transposition reaction products. The products are then introduced into E. coli cells by transformation, and the cells are selected for kanamycin resistance. The colonies scored represent events where ccdB in the target plasmid has been inactivated.Figure 1.

Bottom Line: Transposon-based technologies have many applications in molecular biology and can be used for gene delivery into prokaryotic and eukaryotic cells.If transposition targets ccdB, this gene becomes inactivated, enabling plasmid-receiving Escherichia coli cells to survive and to be scored as colonies on selection plates.The colony formation capacity was linearly correlated with the competence status of the E.coli cells, enabling normalization of experimental data obtained with different batches of recipient cells.

View Article: PubMed Central - PubMed

Affiliation: Division of Genetics and Physiology; Department of Biology; University of Turku ; Turku, Finland.

ABSTRACT

Transposon-based technologies have many applications in molecular biology and can be used for gene delivery into prokaryotic and eukaryotic cells. Common transpositional activity measurement assays suitable for many types of transposons would be beneficial, as diverse transposon systems could be compared for their performance attributes. Therefore, we developed a general-purpose assay to enable and standardize the activity measurement for DNA transposition complexes (transpososomes), using phage Mu transposition as a test platform. This assay quantifies transpositional recombination efficiency and is based on an in vitro transposition reaction with a target plasmid carrying a lethal ccdB gene. If transposition targets ccdB, this gene becomes inactivated, enabling plasmid-receiving Escherichia coli cells to survive and to be scored as colonies on selection plates. The assay was validated with 3 mini-Mu transposons varying in size and differing in their marker gene constitution. Tests with different amounts of transposon DNA provided a linear response and yielded a 10-fold operational range for the assay. The colony formation capacity was linearly correlated with the competence status of the E.coli cells, enabling normalization of experimental data obtained with different batches of recipient cells. The developed assay can now be used to directly compare transpososome activities with all types of mini-Mu transposons, regardless of their aimed use. Furthermore, the assay should be directly applicable to other transposition-based systems with a functional in vitro reaction, and it provides a dependable quality control measure that previously has been lacking but is highly important for the evaluation of current and emerging transposon-based applications.

No MeSH data available.


Related in: MedlinePlus