Getting closer (week 5)

This week definitely had its ups and downs, but I am going to focus on the positives J. I finally have had all four of the plasmids I’ve been constructing come back clean from sequencing! On Monday I had received the news that once again the plasmid pFS1, which was prepared by the highschool intern Felicia, had yet again failed to come back clean from sequencing. Dejected, since I really was hoping I would be successful this time and begin the transformation into Bacillus subtilis with all of my plasmids.   So, we decided to reculture and reconcentrate at a higher level the pFS1-1 strain, and use the 793 down primer instead of the 793 up primer. This was all prepared by Wednesday evening and was sent off to sequencing that night. On Thursday, around 4pm, I was surprised not only with the extremely expedient return with my sequencing sample, but also with what was probably the cleanest and most perfect sample I have seen this entire summer, Awesome! Thus, I was finally able to transform into the wild type the final remaining plasmid deletion strain on Friday! This felt great, and long overdue.
 

Over the rest of last week, I also transformed my two other deletion plasmids (which had come back as clean and good from sequencing Monday morning) into the wild type B.subtilis. By Friday, I had created these wild type strains and extracted their chromosomal DNA for transformation into the mutant strains on Monday. Also, I transformed my whole promoter region DNA into the nsrR, resD & abrB mutant strains (using the chromosomal DNA from the wild type strain I had infected the previous week and extracted last Friday).  By Friday, I was able to freeze these new and completed strains too. This achievement signified that I have successfully generated all of the whole SdpA promoter region strains, and will allow me to begin my first LacZ assay next week :)! This feels like a landmark achievement since this is kinda the place I have building toward this entire internship, and I can’t wait to finally get some results to show for all the work I’ve put in.

Unfortunately, this week I also found out on Tuesday that at some point prior my glycerol stock had been contaminated. Thus I had to re-single clone plate and culture every strain I had previously completed and froze to ensure that none of them were contaminated. By Friday, though, this problem has been taken care of and I am confident that my frozen strains are clean.

This week we also had a guest speaker who enlightend us on what to expect in our academic and professional careers going forward, and how to best plan for ensuring our success. This was very enlightening indeed, since it focused on generating a lot of skills which I had not focused on as of yet - namely, those of networking, leadership, business and marketing. Skillsets which I didn’t really know were so important, well, I kinda knew about the first two, but the last two were a surprise. Definitely something I will have to think about going forward. Also, earlier this week my girlfriend went to Australia, and missed her connecting flight in LAX due to her previous flight running late and them not allowing her to board even though she got to the terminal 30 minutes before her flight left and there were 10 empty seats. Suffice it to say, her being away and all the drama that went on at LAX (she spent the night in the terminal) definitely were large distractions last week, but at least she finally made it there safe and sound J!

I am looking forward to being able to complete the generation of all of the strains I am testing next week, and to begin to finally generate some results via conducting my first, all-be-it a practice run, LacZ assay!