Transcibed by yours truly for you unknown and anonymous readers.

  • iGEM team + Wendell. Team Dicty has about 39 good AB-BC-CD constructs transformed into Dicty and ready/undergoing scrutiny under the microscopes (and eyes). They are still moving 100+ other constructs along our principle pipeline for the creation of parts and constructs.
  • They are freezing stocks of cell lines with mutations in PIP3 and PI3K to study the effects of these mutations.
  • They will also use Angi’s cell tracking program to monitor cell movement in Dicty.
  • Team HL-60 explained transformations, transwells with WT HL-60 and HL-60/GPCR, and FACs/Guava along with the results. hM4D is the RASSL we are most interested in (it has been shown the chemotax well to it’s ligand, CNO).
  • Wendell brings up a good point that because we are putting in so much time and effort into doing this “quick and dirty” assay, there may be better solutions to getting the same data. On the other hand, Aynur has been working every waking hour to help prepare for transwells, which doesn’t leave much room for tweaking the whole process or thinking about other assays.
  • Issues with the transwells came up like the reproducability of results using the same reagents and the same machine as well as where the line is drawn between what fold of response relative to the basal response would indicate chemotaxis. (3 or 4 fold and above would qualify.)
  • I went over how some of the cloning and moving of constructs along the pipeline has shifted to the use of TOPO TA cloning because of wrong primers that give us the wrong attB sites for gateway BP reactions. I also went over the goal of Team HL-60 to fuse a GPCR with a tag at the N-terminus and a movement-specific module at the C-terminus, hence the AB-BC-CD nature of our parts.
  • Katja told us that she is working on optimizing conditions for the cells and the microscopes to be used in conjunction with Angi’s cell tracking program.
  • Hansi’s results from her EZ TaxiScan experiments showed that WT HL-60 respond to fMLP at an optimal concentration of 100 nM and can definately sense a concentration of 12.5 nM fMLP (100 nM/8 = 12.5 nM at the “starting line” of the bridge). She saw high basal activity in HL-60 with the RASSL hM4D and found the optical response concentration to CNO was ~100 nM(?).

Three hour meeting from 10 AM to almost 1 PM. It sure made me hungry by the end. At least we’re all caught up with each other’s work.

So I’ve noticed that I’m getting more sleep deprived every week as I go along and that I start saying yes to everything people in the lab ask or tell me even when I really only half-understand what’s going on. I’m not usually like that, so please try be patient with me if I ask you a question you thought I was clear on what the answer was. I’m working on going back to being more skeptical!

There is plenty to do and plenty to think about, so I will instead invest the next hour thinking about out ethics write-up and reading up on making micro-oxen. Power (I mean macho strength to carry loads, not hierarchial power and physical dominance over other organisms!) to our HL-60.