For these variants, the decreased association rate constants relative to WT suggest that the mutations have a substantial effect on the population of the association-competent fraction of the conformational ensemble representing the M state [37].
DNA replication assay was therefore repeated except that rIciA protein was added to the assay mix before the addition of M. bovis BCG replication competent fraction II.
In-vitro replication competent fraction II was prepared by growing M. bovis BCG Pasteur in 600 ml of 7H9 media supplemented with OADC and casitone, in 1000 ml roller bottle at 37°C to log phase as described previously [34].
The pellet was resuspended in minimal volume of buffer B (fraction II) of around 600 μl and dialyzed for 50 min at 4°C against 1000 fold excess of buffer B. Protein concentration was estimated by BCA and the replication competent fraction was flash frozen in small aliquots, so as to avoid freeze thaw, and stored at −70°C until further use.
For MIV cultures, observed NovRNalR cotransformation frequencies were compared with those expected if all cells were equally competent, (NovR/CFU) * (NalR/CFU), providing estimates of the fraction of competent cells in the culture if competence is a binary on/off cellular state (Goodgal and Herriott 1961; Mell et al. 2011).
That said, Cieplice has a tranquil village charm and offers what seemed to me to be friendly, competent care at a small fraction of what it might cost at an upscale spa.
As we show here, GR containing immunecomplexes obtained with nestin antibodies from the cytoskeleton fraction of anchoring competent cells contained the chaperone HSC70, but not HSP90.
If we link TCR/CD3 distribution to the fraction of cells competent for particle binding, we found that a minimum number of receptors per cell equal to 1.4±0.2×105 was required in order for a cell-particle association event to occur.
The probability that two plasmids are introduced at once into A. marginale organisms could be very low, especially when viability in the extracellular environment might be highly compromised, resulting in a low fraction of cells competent to take up DNA.
The donor segments of these clones were comparable in size, number, and distribution to those of the MIV-selected transformants and included one 'mixed' segment, suggesting that transformation in late-log cultures results from a very small fraction of fully competent cells.
Essentially, this approach represents an in silico substraction procedure, where the proteomes of the non-mycorrhizal species are substracted from the proteomes of the AM-competent species to yield a fraction of the proteome that is expected to be enriched in AM-related proteins.
