Tuesday, October 31, 2006


Problems with the riboswitch assay

This week I got the preliminary riboswitch experiment results. I tested sxy RNA for binding ATP, GTP, dATP. dGTP, guanosine, adenosine, AMP and GMP (separately). Each of these substances was incubated in three different concentrations (10 nM, 300nM and 100uM) with labeled sxy RNA in a special buffer that contains Mg and K. I also had three controls:

1) RNA incubated with 100uM of ligand and not digested with RNase (negative control)

2) no_ligand control, sxy RNA with no ligand added and digested with RNase

3) ladder control, which is sxy RNA digested with RNase under denaturing conditions so that it cuts all of the C and U residues in the sequence (not only single stranded ones).

The results did not seem very exciting at the first glance, since all of the samples gave the same result. Further investigation revealed the assay is actually not working properly. First, all of the samples gave the exact same bandage pattern and the same intensities of the bands. This would be fine and I would have concluded that the ligand addition is not affecting folding of sxy RNA, if only wasn't for my controls. All of my negative controls were digested in the exact same way as the rest of the samples (these RNAs should be intact since no enzyme was added). The very first thing I suspected was RNase contamination. However, if this has really happened we would expect to see more cleavage in the samples where I added RNase (which is not the case).
Another observation was that all of the samples except for the ladder control had very high background, meaning that there is lot of unspecific cleavage (that gives fragments of various lengths and resolves as darker background in a gel).

One of the explanations of what happened is that the buffer that I used for riboswitch analysis was somehow affecting RNase activity or (and) unspecifically digesting the RNA. The unspecific digestion of RNA usually happens when the solvent is too basic. I checked the pH, concentration and composition of the riboswitch buffer and it is not much different in composition from the regular buffer (the one that I use for secondary structure probing). The only thing that is different is pH - the riboswitch buffer is more basic (pH 7.9) than the regular one (pH 7). According to the technical support that I called, this shouldn’t make much of a difference for RNase activity however, I suspect that this is the major problem in the assay. I did bunch of controls yesterday testing all of the buffers and reagents that I use in the experiment and ran the samples in a gel. I should have the answer of what is going on later today. If it turns out that the riboswitch buffer is the one to blame, I think I will try doing the assay using the regular buffer for riboswitch analysis.

Another news is that I got my thesis back form all of my committee members and I am currently working on the revisions. This shouldn’t take too long and I think that I‘ll have everything done and ready to go by the beginning of next week.

Monday, October 23, 2006


Research status

In last couple of weeks I handed in my thesis (finally!), I started working on the riboswitch experiment and made a nice poster for the LSI research day.


I finally finished it!!! The last week of writing was pretty rough: I slept for only couple of hours in the full week. I it took me a full day to finish my table of contents and list of tables and figures (somehow I always thought that those are the stuff that could be done more as a side work). The only thing that my thesis is sill missing is an abstract, which I’ll have to finish this week. Currently, I am waiting to hear back from my committee members.


LSI grad research day is taking place tomorrow it seems that is really nicely organized. I am going to present my poster in the afternoon. Actually, the poster is designed to present combined data on regulation sxy expression where we included the results from most of the work on sxy. The poster session starts tomorrow at 330pm. All are welcome!


This is experiment that I started some time ago. The idea was to test whether sxy mRNA binds small molecules, especially purine bases, nucleosides or nucleotides. Traditionally this is done via in-line probing, which is based on the idea that RNA spontaneously degrades (incubation at 25C in salts) predominantly in single stranded regions. The end labeled RNA is incubated with and without ligands, and resulting fragments are then resolved on sequencing gels. If the RNA binds the ligand the RNA secondary structure changes and this can be detected in the band pattern change.
However, the in-line probing did not work for me and we decided to use alternative way to test sxy RNA by performing nuclease mapping assays. This is similar to the previously described one where the only difference is use of RNases (that specifically cut certain residue type) to degrade RNA.
Since there is so many different ligands that we could test (and both time and money are limited!) I am going to focus on testing nucleotide tri- and mono- phosphates first.
Here is the logic – H. influenzae cells can gain nucleotides in two ways:

1) Through DNA uptake
where DNA is degraded and the dNTPs are reused

2) and by taking up free nucleotides from their environment. However, cells cannot transport nucleotides unless they are first dephosphorylized to give nucleosides. This happens in periplasm and up taken nucleosides are then converted in nucleotides and used in the cell.

Therefore we would expect that the signal for sxy expression is probably one of the purine nucleotides or nucleosides (this also includes IMP because AMP interconvert with GMP through IMP).

I prepared labeled sxy RNA and ligands that I want to test, and I am going to do the first tests today. This week we should have some interesting (hopefully) results.

Tuesday, October 03, 2006


Always look on the bright side of life..

Ok, I am closer to the beginning of the end with my thesis writing.
I admit that it makes me really happy the fact that I have to use an industrial stapler to staple my thesis together. Also the Rosie's comments are getting much shorter and there is more "good"s on the sides of my poor paragraphs.
I think that these are all the signs that I am getting there..

After two sleepless nights, finished my second draft of discussion section today. I am really unhappy with it but I am ready to work on it for the rest of the week knowing that soon it will be completely done. My goal is to finish drafting the thesis by the end of the week. (!!good luck thesis!!)

End now for something completely different-

While I was doing some background reading, (in my pathetic attempt to describe one of the models of sxy regulation in my discussion) I found the data for nucleotide concentrations in the cells of E. coli and it was quite interesting. It turns out that NMPs are far more abundant in the cell than the NTPs, and that GMP is found in much higher concentration in the cell than the rest of the NMPs. Knowing that AMP and GMP can interconvert through a common precursor, IMP and that the conversion is especially important when guanine and adenine compound are present in medium (and available for the cells), the data seems surprising. It would be interesting to find out why this NMPs ratio differs.

Does anyone have an explanation?

Tuesday, September 26, 2006


Very first thesis draft

Last week I have been working on writing of the discussion section and preparation of my first thesis draft. The discussion section is still not fully developed but I got a very basic first draft.

Additionally, it was my turn last week for lab meeting, so I turned it into discussion on my thesis discussion. I presented some ideas on the regulation of sxy genes. Based on the information that we have so far I proposed that sxy could be regulated by one of these three mechanisms: transcription/ translation coupling, action of a riboswitch or by binding of non-coding RNA. At this point it seems that sxy regulation can be explained by each of these mechanisms however, there are also some problems and none of this models fits perfectly into the picture. I think that I’ll still have to do some reading on this subject (and maybe to discuss it with my lab mates:)).

Yesterday I revised the intro and methods sections and I think that they got much better. Today, I am starting with the revision of my results section (which at this stage has quite a bit of problems). The goal for this week is to have a nice readable draft with all of the figures and references included.

Monday, September 18, 2006


discussion on my thesis discussion

Last week I finally finished the first draft of the introduction and handed it to Rosie. I am still not very happy with it but at least it is a start and I'll work from that.
I think that I have a problem to make a distinction between important stuff and details. Additionally, I find it really hard to make up a story that is interesting and easy-to-follow (than again, maybe science is not supposed to be told in an interesting way?). They say it comes with time and practice, but I do not have either.

Today I started working on the discussion section. I made a very rough outline and realized that I 'll have quite a bit of reading to do to be able to propose the regulation of sxy gene. I have some ideas how to organize this section and what kind of arguments to make.

Tomorrow is my turn to give a lab talk, so I'll have a busy night preparing the presentation on my discussion (which I still do not have). I am kind of looking forward the presentation, hoping that I'll get feedback that will improve my analysis and give me new ideas.

Tuesday, September 12, 2006


still working on the gel picture........

I ran some gels last week and it turned out that the label (on the RNA) was really old so I had to expose them for days and days to be able to visualize the bands (and they were still fuzzy). I ran another gel today and I will have to leave for at least 4 days to get something (I am getting really annoyed with it).

I finished the first version of the gel figure that we want to include in the sxy manuscript. I decided that it is not the_worst_thing_ ever to crop your gel picture and to show only the lanes that carry important info. It is a bit of a patchwork, but I think that it doesn't look that bad.

Thesis writing is going very slowly. I have to do some background reading to improve my introduction info. Hopefully, I will have this section done (together with Methods and Results section) in couple of days. Then, I can finally start working on the discussion!!

Thursday, September 07, 2006


Back to lab

After couple of weeks of sitting at my desk and trying to write my thesis, I decided to run more sequencing gels. Why? - you ask.

Our lab is currently preparing a manuscript on regulation of sxy gene in H. influenzae and we would like to present the data showing that sxy RNA secondary structure limits the sxy expression.
One way of indirectly showing this is to do the mapping of the secondary structures of RNAs in the wt sxy and of RNAs of sxy hypercompetent and non (or hypo) competent mutants. Both of these mutants carry point mutations that affect the stability of their RNA secondary structures.

The analyses that I did so far confirm that the secondary structure of sxy RNA is less stabile in the hypercompetent mutant and more stabile the hypocompetent mutant. Eventhough I got nice and reproducible results (with nuclease mapping of secondary structures), I still do not have the one -? perfect and publishable picture.

This week I will try to produce a nice gel picture showing the differences in the sxy RNA secondary structure in these different RNA species. Although this sounds as a really simple thing to do, I can easily assure you is highly stressful procedure!
Yesterday I prepared the RNA samples and today I am running them in a gel. Hopefully we'll get some very nice results tomorrow!!

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