Description:

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. 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 MUT-849, however the wild-type cell-walled strain MUT-125 is as sensitive to tetracycline as strain MUT-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.

Bacterial Host Strain:

DH5 alpha

Selectable Marker:

Amp-r