Subategory:

Mutants

Description:

The B12 background strain is MUT-1820 (arginine auxotroph) into which a genomic insert has been integrated (location not determined) that encodes the following: an intact ARS2 (arylsulfatase, expressed constitutively) cassette; an intact ARG7 cassette (to select transformant by restoring arginine synthesis); a ~1600 bp intron; and the 3' half of the coding sequence of the Hygromycin resistance gene, followed by a 3' UTR. This construct is available as plasmid pHR18. Parental strain MUT-1820 was transformed with pHR18 by electroporation and colonies were obtained by selection on TAP plates without arginine added. Integration of the full 3' end of the construct was screened by PCR, and then candidate colonies were screened by arylsulfatase assays to screen for integration of the 5' end as well as to determine that the locus was well expressed. Strain B12 tested positive in both of these assays. B12 grows in regular TAP media, or in HSM; it does not require supplementation as its arginine prototrophy has been restored. This strain is designed to screen for homologous recombination (HR) by transforming it with plasmid pHR23. Upon homologous recombination of pLP23 with the pLP18 locus, the cells become resistant to hygromycin by reconstituting the hygromycin resistance cassette. A paromomycin resistance cassette in pHR23 allows for a measure of the total transformation efficiency, such that the ratio of HR to non-homologous integration can be determined from a single transformation.

Species:

Chlamydomonas

Locus:

ARG7

Chromosome:

1