It’s all kind of surreal, the fact that I’m leaving iGEM and starting college. It’s an exciting time in my life. I’ll do my best to balance school, iGEM and fun. I’m going to miss everyone in the Lim lab. Look out for when I come back!
Bloggin’ out. Will return for more iGEM.
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.
Label your food. Or else Aynur will eat it!!!
:) Just kidding. I love Aynur. It was pretty funny how she ate David’s sandwich.
I am behinddd! We grew cultures overnight yesterday hoping to do minipreps and digests today, but we forgot to add antibiotics to our media. This sucks. How many times does it take for me to forget to add the stuff before I remember to? Oh boy. Today was a mix of picking/growing new colonies and transforming/re-transforming plasmids. It’s basically what Eric and I have been doing all week! MINIPREP MANIA TOMORROW.
Peasant Pies sells interesting drinks.
Crayon drinks. Cool, eh?
Right now, I’m just sitting in the cave waiting for Eric and Katja to do 26 minipreps for 13 successful transformations. We re-did transformations for 8 constructs, but they still need incubation/growing time on CARB or KAN plates. We will do minipreps for those 8 tomorrow.
In other news, it’s iGEM week 3. I met my buddy, Jason, who is in the joint graduate student group between UCSF/Berkeley. He’s on track to get his M.D./Ph.D. dual degree. Team HL-60 is split up between Anyur and Jason/Bethany. Anyur is teachcing Cathy and Rye-lye how to perform the transwell assay while Jason and Bethany are teaching Eric, Katja and me how to transform TG1 cells (strain of E. coli) with plasmids contianing our constructs.
This morning, we also went to bug Arthur to teach us how to operate the EZ Taxis Scan machine. I have to go back there and finish assembling the assay. Speaking of which, I left my pilot pen in the Weiner Lab. Gotta go get it in a couple minutes.
Bethany was saying how team HL-60 doesn’t have post-it posters with agendas and constructs posted up on the wall for easy reference. Maybe we should buy crayn drinks and draw on posters with crayons. Hah. Cheesy. Blog again later!