I want to test whether competent cells will bind to DNA that's attached to the surface of beads. I'll use magnetic beads for this, rather that the polystyrene beads I'll use with the tweezers, as I can easily separate them from cells. In principle I just need to mix cells and beads, pull out the beads, and plate them to count the number of beads that have attached cells But the details need some thought.
In fact I already gave this experiment some thought, but I was derailed by finding that the beads formed big clumps when mixed with competent cells and by running out of competent cells. I found that the BSA-coated ('MyOne') beads didn't clump so I'll use these, even though they're not the ones recommended for DNA work. And I've made new cells. But I didn't think much about some other details.
One is timing. I think I should allow only a short time for the cells to interact with the beads. This is more critical for B. subtilis, which can cut bound DNA and so might break lose of the beads. So I think I'll give the H. influenzae cells 5 min to find the DNA, and the B. subtilis cells only 1 min. Then I'll chill the tubes on ice before exposing them to the magnet.
Another issue is the absolute and relative concentrations of cells and beads. I think I should use similar numbers of cells and beads. My frozen competent H. influenzae cells are at about 10^9 cfu/ml. The frozen competent B. subtilis cells are more dilute, ~2x10^8/ml. The MyOne bead stock is even more concentrated, ~10^10 beads/ml, but I only have 1.0 ml and will use only a little bit of this.
So let's say I coat 10 µl of beads with DNA, and mix them with 100 µl of H. influenzae cells, and after incubation I dilute this to 1 ml with cold wash solution. Should I plate both the bead fraction and the supernatant ('side-natant'? call it the SN) fraction, and how many washes should I do? If all the cells are competent and all the beads are DNA-coated, then all the cells might bind to the beads. If most of the cells aren't competent, or if cells are inefficient at finding and binding to the beads, most of the cells will be in the SN, and plating it will probably not be sensitive enough to detect the small decrease.
Let's conservatively assume that only 10^6 cells bind to the 10^8 beads, and that about 5% of each SN is carried over with the beads in each wash. The first SN will still contain ~10^7 cells/ml, and the bead fraction will have 1.5x10^6 cells (~10^6 attached and ~5x10^5 free). I'll resuspend the beads in 1 ml, mix gently, and collect the bead fraction again. The second SN will contain about 5x10^5 cells/ml, and the beads will contain about 10^6 (~10^6 attached and 2.5x10^4 free). If I wash these beads a third time, the SN will contain about 2.5x10^4 cells/ml and the bead fraction will still contain about 10^6 cells.
So I could get away with doing only two washes, but doing a third would give more confidence that the cells are really attached. This assumes that the cells stay attached to the beads through the washings - this will require mixing them quite gently and keeping them cold, and hoping.
One other complication is that counting the cells on the beads uses up beads. But I don't want to count more than ~100 colonies, so I could just take 10 µl of each 1 ml resuspended bead fraction for dilution and plating.
Welcome to the 4th Reich part 1.
4 hours ago in Angry by Choice