Few poets have been lavished with greater gifts than Walcott; but much of his later work has been unadventurous (and undistilled), full of stock passages and stale opinions.
All cultures were initiated from a cryopreserved glycerol stock, passaged twice for growth uniformity and grown until the mid-exponential phase at 37°C (actively dividing stage of bacterial growth) prior to the SPOTi assay.
These stocks (Passage 4) typically contain about 1x108 pfu/ml.
The other cultures were all evolved for 1000 generations (100 passages), except for line A2 which got contaminated after 33 passages, but was restarted from the frozen stock at passage 30 and was evolved for 820 generation (82 passages).
For microarray analysis, FE1 cells were propagated from one vial of cryo-preserved stock of passage #19.
For the cell culture model, BGM cells with EMEM culture media without antibiotics (10%% bovine serum albumin, 1 % NEAA, 1 % glutamax) were used to propagate C. burnetii from stock cultures (passage n = 10 12 in BGM cell culture) until stationary growth phase [ 20].
However, Western blotting analysis of Vero E6 cells infected with AUG 1 KO virus from either the original stock or passage 5 showed that P78 could be expressed, albeit to a lower extent, from the newly introduced initiation site (Supplementary Figure S1B), suggesting that the in-frame stop codon is too close to the initiation codon to be recognized.
The virus stock (2 passages in Vero 76 African green monkey kidney cells) used was from a clarified cell culture lysate preparation concentrated using an Amicon stirred ultrafiltration cell (Millipore, Danvers, MA).
Nevertheless, high constant production of FTM- proteins was achieved by preparing large stocks of passage 3 that were then used to inoculate repetitively eggs for protein purification from passage 4 (see Figure 1C).
