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Begining to infect my B.subtilis & some frustrations (week 4)

This week has been a mixed bag.  It started out well.  I got to begin by learning how to transform the Bacillus subtilis and by inserting my whole promoter sequence (which came back successfully two weeks ago from sequencing) into the wild type strain of competent cells I generated last week.  This involved making DSM agar with Erythromycin as the antibiotic resistance selection agent (since the plasmid will give this resistance to the B.subtilis which it infects).  When I came back on Tuesday, however, I arrived to the unpleasant surprise of having colony growth on my control plate, BOO!  Thus I had to continue forward as though this didn't mean the contamination would ruin my experiment while at the same time preparing a new strain of competent wild type B.subtilis cells as a back-up plan if this doesn't work out.  The probable reason for this source of contamination is due to that when you grow wild type competent cells you have to do it in media which doesn't have any antibiotics since there are no knockout genes which confer these cells with an associated antibiotic resistance - thus you have to be VERY careful to avoid contamination when you make them.  Thank-fully when I verified the cells on TSS limited media plates it showed that the insert did take place as planed and the contamination probably didn't ruin my results. I know this because these plates have different amounts of the required amino acids: Phe, Trp & Thr and without a necessary amino acid they will not grow (Thr being the one which we eliminated the ability for the cells to generate biosynthetically because we inserted via homologous recombination into a gene required in its biosynthetic pathway and thus they would not survive on plates without this supplied in the media).

Also, this week I have been awaiting the sequence data we sent out to the primate center last Friday.  We cannot transform the other wild type cells with the deletion segments till we verify they have the correct sequence and no mutations took place.  Unfortunately, even though we sent them to the center last Friday, we didn't get them back till this Friday, and thus had very little to do in the lab some days last week.  Then to add insult to injury, the two sequences I sent both seemed to be contaminated and will need to be sent again (though when I called them, it seems that this could just be due to them not being concentrated enough to avoid the background noise which is pretty much always there).  Thus I reconcentrated new isolates of these segments at higher concentrations and got them ready for sequencing again.  Hopefully I get them back more quickly this time and they are all considered good :)

On Wednesday this week we had a brown bag seminar on science ethics, and it was quite interesting!  I have had classes like this before, but they centered more on whether or not to conduct certain types of research, how this research should be conducted and avoiding making up results.  This time, however, it centered entirely on the ethics involved with publishing your results or just writing about them in general.  Wow!  I had no idea this was such a complex quagmire of issues, in which one can quite quickly and easily get oneself into Extreme trouble.  I'll definitely be MUCH more careful in the future with these types of issues.  Very useful tips indeed.  Though to be honest, in many respects it left me a little confused about what the lines actually are, since we didn't come up with any such definitions, but instead just generated more questions.  

This Friday I got the chance to present my preliminary results to the other interns and frontline mentors.  This was a little difficult for me since I have not been able to generate ANY results as of yet, since I have spent this whole time generating my mutants.  Also, we needed to sum up our 4 weeks worth of work into 5min and up to 2 PowerPoint slides.  I tried my hardest to fit into these constraints, though when I presented I saw that these weren't as strict of guidelines as I thought.  I would definitely have liked to have had more slides which made my work more itemized and easily to understand (thus flow better).  As it was, I did get it into two slides, but they weren't the most helpful for generating flow, since they were super-packed with information, and I had to skip between them a little bit.  Also, I completely forgot to mention (though it was written in my slides) that my project was being conducted in-vivo and why this is so important to what I am investigating.  Ah, well.  I definitely will do better with my final presentation.

So yeah, that was my week.  Ups and downs, but it seemed like more downs than ups.  At least I was able to get the chromosomal DNA isolated for next week’s transformations into the mutant competent strains.  Next week, I am thus hopeful that I will be able to begin to do some LacZ tests so I can begin to generate some results at long last!