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Literature summary for 2.7.7.B22 extracted from

  • Thakker, C.; Lin, K.; Martini-Stoica, H.; Bennett, G.N.
    Use of transposase and ends of IS608 enables precise and scarless genome modification for modulating gene expression and metabolic engineering applications in Escherichia coli (2016), Biotechnol. J., 11, 80-90.
    View publication on PubMed
Search for more literature for 2.7.7.B22

Cloned(Commentary)

Cloned (Comment) Organism
IS608 encoded transposase gene tnpA, DNA sequence determination and analysis Escherichia coli O103:H2

Protein Variants

Protein Variants Comment Organism
additional information development of a simple and precise method for genome manipulation in Escherichia coli that alters the gene sequence without leaving foreign DNA in the chromosome. This strategy involves PCR amplification of a DNA cassette containing ISHp608-LE (left end)-antibiotic resistance gene-counterselection marker-ISHp608-RE (right end) by using primers containing extensions homologous to the adjacent regions of the target gene on the chromosome. The lambda Red-mediated recombination of the PCR product and antibiotic resistance screening results in transformants with a modified gene target. The ISHp608-LE-antibiotic resistance gene-counterselection marker-ISHp608-RE cassette can then be excised using a temperature sensitive plasmid expressing the TnpA transposase, which precisely cleaves ISHp608-LE and ISHp608-RE without leaving a scar sequence. For introduction of IS608 LE and RE into the gene of interest, lambda-Red recombination is utilized, which does not require in vitro manipulations such as restriction digestion, ligation or construction of a suicide vector. Diagram of plasmids containing selectable and excisable IS608 cassettes, overview Escherichia coli O103:H2

Localization

Localization Comment Organism GeneOntology No. Textmining
nucleus
-
 Escherichia coli O103:H2 5634
-

Organism

Organism UniProt Comment Textmining
Escherichia coli O103:H2
-
-
-
Escherichia coli O103:H2 EHEC
-
-
-

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
additional information the TnpA transposase precisely cleaves leaft and right ends (LE and RE) of a gene without leaving behind a scar sequence. Development of a simple and precise method for genome manipulation in Escherichia coli that alters the gene sequence without leaving foreign DNA in the chromosome. This strategy involves PCR amplification of a DNA cassette containing ISHp608-LE (left end)-antibiotic resistance gene-counterselection marker-ISHp608-RE (right end) by using primers containing extensions homologous to the adjacent regions of the target gene on the chromosome. The lambda Red-mediated recombination of the PCR product and antibiotic resistance screening results in transformants with a modified gene target. The ISHp608-LE-antibiotic resistance gene-counterselection marker-ISHp608-RE cassette can then be excised using a temperature sensitive plasmid expressing the TnpA transposase, which precisely cleaves ISHp608-LE and ISHp608-RE without leaving a scar sequence. For introduction of IS608 LE and RE into the gene of interest, lambda-Red recombination is utilized, which does not require in vitro manipulations such as restriction digestion, ligation or construction of a suicide vector. Diagram of plasmids containing selectable and excisable IS608 cassettes, overview Escherichia coli O103:H2 ?
-
 ?
additional information the TnpA transposase precisely cleaves leaft and right ends (LE and RE) of a gene without leaving behind a scar sequence. Development of a simple and precise method for genome manipulation in Escherichia coli that alters the gene sequence without leaving foreign DNA in the chromosome. This strategy involves PCR amplification of a DNA cassette containing ISHp608-LE (left end)-antibiotic resistance gene-counterselection marker-ISHp608-RE (right end) by using primers containing extensions homologous to the adjacent regions of the target gene on the chromosome. The lambda Red-mediated recombination of the PCR product and antibiotic resistance screening results in transformants with a modified gene target. The ISHp608-LE-antibiotic resistance gene-counterselection marker-ISHp608-RE cassette can then be excised using a temperature sensitive plasmid expressing the TnpA transposase, which precisely cleaves ISHp608-LE and ISHp608-RE without leaving a scar sequence. For introduction of IS608 LE and RE into the gene of interest, lambda-Red recombination is utilized, which does not require in vitro manipulations such as restriction digestion, ligation or construction of a suicide vector. Diagram of plasmids containing selectable and excisable IS608 cassettes, overview Escherichia coli O103:H2 EHEC ?
-
 ?

Synonyms

Synonyms Comment Organism
ECO103_3558
-
 Escherichia coli O103:H2
IS608
-
 Escherichia coli O103:H2
TnpA 155-amino acid transposase
-
 Escherichia coli O103:H2
TnpA transposase
-
 Escherichia coli O103:H2

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
30 37 assay at Escherichia coli O103:H2