OK, the draft NIH proposal is in the hands of the internal reviewers. Early in January they'll give me lots of (I hope harsh) feedback on what needs to be fixed. Till then, I've got two big things on my plate.
One is the US variation manuscript. It isn't exactly on my plate yet, as my coauthors are still assembling their drastic revisions back into a coherent manuscript, but they say I should get it tomorrow. I'm hoping it's now in pretty good shape, with my job now just to polish the writing and the figures so we can get it submitted within a few weeks.
The other is beginning the groundwork for the optical tweezers experiments. Let's see what I can remember about what needs to be done. In general, I'm going to use Bacillus subtilis cells as my positive control, because they're bigger and tougher than H. influenzae. They're also naturally competent, and were successfully used for an optical tweezers study of DNA uptake in 2004.
1. I need to be able to stick cells onto glass coverslips without killing them or otherwise preventing them from taking up DNA. I'll start with Bacillus subtilis, and with poly-L-lysine coated cover slips. I'll need to make these myself - I have several protocols and several offers of help, but don't know if any of these people will be around over the holidays. The alternative to poly-L-lysine is a silane solution (fancy name) that was used for the B. subtilis experiments. But I don't have a protocol for using this, so it's a bit of a crapshoot. Some of the poly-L-lysine protocols say to pre-clean the coverslips with strong acid (nitric? chromic?) - a researcher down the hall said he might have some (old-tyme labs are good to have around).
2. I need to attach streptavidin to polystyrene beads. I have a protocol, and the streptavidin, and the coupling reagent, and the ready-for-coupling beads (they may be a bit small, 1 µ rather than 2 µ, but they'll do). What I don't have is a good way to test how well the coupling has worked (see below).
3. I need some biotin-conjugated DNA (H. influenzae chromosomal DNA). The research associate made some a while back for a different experiment, but I don't know if there's any left, or where it would be. I could make my own, if I can find the biotin.
4. I need to make the B. subtilis competent. This means that I need to make up the appropriate culture medium and competence-inducing medium (2 kinds, as I recall), and the appropriate agar plates for selecting transformants (so I can test whether they really are competent).
5. Once I have the streptavidin-coated beads and the biotin-coupled DNA, and some competent cells, I can test whether cells will stick to beads that have been incubated with DNA but not to beads without DNA or to DNase-treated beads. If this works I will know that there's streptavidin on the beads and biotin on the DNA and the cells are competent. If it doesn't I'll only know that at least one thing isn't right.
6. At this stage I can also test whether the cells I've stuck onto a coverslip can still bind DNA, by giving them the beads-plus-DNA and seeing if the beads stick to the cells (with the same controls as in step 4). Oh, but first I have to make sure that the competent cells will also stick to the coverslips.
7. Then I can make some competent H. influenzae and try steps 5 and 6 with them. Assuming I've been able to stick the H. influenzae cells onto coverslips).
8. After all this is working, I'll be ready to go back to the physics lab and try to measure some forces!
20 hours ago in The Phytophactor