It's all about the multitasking (week 3)

I had no mistakes which needed redoing this week! That was a super plus. Also, I amazingly got back my sequence by Monday morning (we didn’t think we would be getting it till like Thursday), and it showed that I had a perfect plasmid (not the case with Keifer – the high school teacher). So for me, that meant I am ready to transform this plasmid into Bacillus subtilus next week.  Before I can do that, however, I needed to make my B. subtilus competent cells for the wild type and each of the single mutants I am working with (for me this was ResD, NsrR & AbrB, and Keifer also had Fur instead of AbrB). Also, since I had a good plasmid come back from sequencing, it meant I could begin to try to make up for last week's unsuccessful attempt to use the 194/192 primer set to generate the missing promoter deletion sequence.  This time using the wild type plasmid DNA, which has the whole promoter sequence, as the template instead of the host DNA.
 

Thus I found myself in the interesting position of doing procedures from both week 1, 2 & the new stuff from this week concurrently. The best of all, I didn’t have any major screw-ups! Well, at least as far as I know.  By the end of the week, I have gotten the 194/192 deletion to the single clone stage (which means I will have it ready for sequencing by Tuesday hopefully), have finished getting the 195/192 deletion to sequencing and am awaiting the sequencing data, and have generated B. subtilus competent cells for the wild type, NsrR, ResD, AbrB & Fur. Very busy week indeed. Making B. subtilus competent cells turned out to be a very time consuming process (all together takes around 8 hours, but there are around 6 hours where you do nothing but wait for stuff to finish incubating), so it is all about trying to get as early of a start as possible if you want to leave at a reasonable time. Also, the Fur mutant cells didn’t plate very well on the antibiotic resistance plates, so we had to wait till Friday (after re-plating those that did grow on Thursday) to make these competent cells.
 

Also this week we had a VERY interesting guest speaker on Fridaywho spoke of how her career path was FAR from what one would expect (especially since she has been SO successful at various times throughout her career, only to have to restart on many occasions). Very interesting perspective indeed, and eye-opening that I shouldn’t expect any plan to really work the way I hope and to be open to all that life sends my way. One way or another, everything I do will combine to create my skill set and alter my future, an often in ways I never expected.
 

I am excited that next week I will finally begin to finish creating the final mutants I am conducting my study on. Hopefully I will be able to begin generating results sometime during week 5!