AtetX
$30.00
From Sergio García and Guy Cardineau, Tecnológico de Monterrey, Campus Monterrey-Mexico, May 2015
The construct was generated by Polymerase Chain Reaction, amplifying the open reading frame of tetX from the BtetX plasmid with a proof-reading high fidelity (1 error/100,000 bp = 0.001%) enzyme and oligos that carried XhoI and BamHI sites in their 5’ends. The amplicon was digested with BamHI and XhoI, and cloned into the corresponding sites of plasmid pHsp70A/RbcS2-cgLuc, replacing the luciferase ORF with that of tetX.
Comment: The AtetX plasmid can be used to select nuclear transformants of Chlamydomonas reinhardtii. It has been proven to work in the cell wall deficient strain CC-849, however the wild-type cell-walled strain CC-125 is as sensitive to tetracycline as strain CC-849. The transformants should be selected in in TAP plates containing 15 µg/mL of tetracycline and maintained below a light intensity of 24 µmoles m-2 s-1. Transformed colonies will be visible after 8 days and can resist up to 100 µg/mL of tetracycline.
host strain: DH5α
amp resistant