Field of Science

Uptake sequence manuscript done, back to the tweezers experiments

Yesterday I finished revising all the components of our manuscript on uptake sequence evolution, and sent them to the co-author who's handling the resubmission.  it was already a good manuscript, but now it's very very good, so if the editor and reviewers don't like it we'll just sent it somewhere else.

I've been working on the components of the optical tweezers experiments, with modest progress.  The tweezers apparatus still had problems when I was there the other day (it wasn't aligned properly?), but I have lots of other parts to work on.  Here's a list:
  • The RA of the tweezers lab showed me a way to pre-clean coverslips, and I need to test the ones he prepared to see if they give more consistent results than the ones I've been using. (Coat his and mine with poly-L-lysine, make into chambers, test binding of competent cells.)
  • I need to test whether washing treated coverslips with culture medium (BHI) or protein (BSA) blocks cell and bead binding, because once I have cells bound to the surface I don't want beads to stick to it.
  • I need to make a big batch of competent cells and freeze them in 0.5 ml aliquots, because the cells I made before were contaminated.  (The contaminants were flagellated cells that otherwise looked just like H. influenzae - they were the spinning wiggling cells I described in my last post.)
  • I need to test whether cells that have stuck to a coverslip are still able to grow.  To do this, my plan is to replace the medium in the chamber with medium containing low-melt agarose, and once this has set incubate the chamber at 37 °C for a few hours and then examine the cells under the microscope, looking for clusters of cells (microcolonies).  As a control I'll first try this with cell-free chambers and agarose medium that already contains growing cells.
  • For today I'm just going to work with H. influenzae, but I should also test these factors with B. subtilis cells.

1 comment:

  1. If you give me a few days' warning of when you're next coming, I'll try to get into the lab and realign the tweezers myself for you!


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