The cytosine deaminase negative selection system
This page provides vector sequences and advices facilitating the use of the cytosine deaminase negative selection system
for gene disruption and replacement in streptomycetes and other actinobacteria.
Official publication: Dubeau, M.-P., Ghinet, M. G., Jacques, P.-É., Clermont, N., Beaulieu, C., Brzezinski, R. (2009)
Cytosine deaminase as negative selection marker for gene disruption and replacement in the genus Streptomyces and other actinobacteria. Applied and
Environmental Microbiology 75:1211-1214. Read the abstract. (read an extended abstract)
Properties of the vectors:
All the vectors of the pMP series include the wild type cytosine deaminase enzyme (from E. coli).The gene, codA(s) has been synthesized de novo, to adapt the codons
to expression in actinobacteria. [sequence link] The vectors of the pMG series include a mutated version
of this gene, codA(sm). The mutated protein is characterized by an enhanced affinity to fluorocytosine compared with cytosine.
We recommend the vectors of the pMG series, as they require lower concentrations of 5-fluorocytosine for selection.
All the vectors include an origin of replication for E. coli (ColE1-type) and two polylinkers.
"201-type" vectors include a kanamycin resistance gene (from E. coli transposon Tn5). They will be used for procedures in which DNA is introduced in Streptomyces by protoplast transformation or
"301-type" vectors include a kanamycin resistance gene and an origin for conjugative transfer (oriT). They will be used in conjugative procedures, when the E. coli donor itself is not resistant to kanamycin.
"302-type" vectors have been constructed to be used in conjugative procedures with popular E. coli donors which are resistant to kanamycin. For this purpose, the vectors include an additional ampicillin-resistance
We recently constructed the "303-type" vector (unpublished). Like the other "3xx"-vectors, it includes ori(ColE1), codA(sm) and oriT, but it has the apramycin-resistance determinant. It can be used in conjugative procedures
involving popular, kanamycin-resistant E. coli donor strains, but it is smaller than the "302-type" vectors.
Retrieve vector sequences:
pMG201M vector (raw sequence) (annotated MS Word file) (Map)
pMG301M vector (raw sequence) (annotated MS Word file) (Map)
pMG302M vector (raw sequence) (annotated MS Word file) (Map)
pMG303M vector (raw sequence) (annotated MS Word file) (Map)
Recently, a spontaneous mutation have been noticed by DNA sequencing in the plasmid pMG302M (Nancy Allard & Sébastien Roy, personal communication).
The mutated nucleotide is localized immediately after the -35 box of the promoter of the codA(sm) gene. The mutation could be present in other vectors as well.
Retrieve the sequence of the pMG302M vector with the mutated nucleotide:
pMG302M vector with mutation (annotated MS Word file)
The vectors of series 201, 301 and 302 have been constructed by Marie-Pierre Dubeau and Mariana Gabriela Ghinet. The 303-series vector has been constructed by PhD candidate Mebarek Lamara.
Other details will come soon. This page is in development.
Video: Protoplast preparation and transformation