I've done several more tests of how cAMP and the presence of inosine inluence competence development. All these experiments used wildtype cells and the 'mixture' medium RM. In each case I grew the cells in RM plus inosine for at least several hours at low density, so that competence was very low. I then transferred them to different media that I thought would affect their competence (keeping the cell density about the same), and took samples.
Here's a graph of the results of the latest experiment. The bottom (blue) line is the negative control: cells were put into fresh RM medium with inosine. They continued to grow and became a bit ore competent as the culture got denser, but competence was still much lower than that of dense cultures. The top (purple) line is the positive control: cells were put into the starvation medium MIV, which caused them to become very competent very quickly. As I recall, cells transferred from rich medium to MIV take longer - I should look up some data.
The red line is cells transferred from RM plus inosine ('RM+') to RM minus inosine ('RM-'). The cells can still grow in this medium (with a variable lag), but there's a rapid (though modest) increase in competence. (Hmm, I've been thinking that this competence is much higher than that of cells that were growing continuously in RM-, mainly because that was true the first time I did this transfer. But I need to check my data for continuous growth in RM-, and maybe do more controls.)
Cells became tenfold more competent if they were instead transferred to RM+ with cAMP (teal line), and the combination of RM- and cAMP induced even higher competence (green line), up about 1000-fold from cells in RM+. But this is still 100-fold lower than the MIV-induced competence. How come?
The difference is probably because of something that RM- has and MIV lacks. It shouldn't be the amino acids from casamino acids, as both media contain these (MIV has less), but RM has a lot of other ingredients that come from the RPMI component. I wonder how much competence would be induced by transfer from RM+ to cMMB without inosine but with cAMP? Perhaps I'll do a series of tests of adding different RM components to MIV.
Why I'm Marching for Science
6 hours ago in Angry by Choice