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.
CHAPTER I - THESIS
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 IÂll have to finish this week. Currently, I am waiting to hear back from my committee members.
CHAPTER II - POSTER SESSION
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!
CHAPTER III - RIBOSWITCHES
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.
CHAPTER I - THESIS
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 IÂll have to finish this week. Currently, I am waiting to hear back from my committee members.
CHAPTER II - POSTER SESSION
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!
CHAPTER III - RIBOSWITCHES
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.
# posted by Okapia Johnstoni @ 9:37 AM 
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One of the nice things about using the "outline" feature of Word is that it will automatically generate a table of contents for you. At least it used to do that.
If you haven't already set up the outline, or if you want to change it, hte Faculty of Grad Studies used to have (probably still does) a Word "template" file for theses that sets up the formating of the headings and text. It's designed for PhD theses but should also work fine for MSc theses.
If you haven't already set up the outline, or if you want to change it, hte Faculty of Grad Studies used to have (probably still does) a Word "template" file for theses that sets up the formating of the headings and text. It's designed for PhD theses but should also work fine for MSc theses.
It seems inefficient to dephosphorylate nucleotides in the periplasm, only to phosphorylate them again in the cytoplasm. I would be interested to know why this has to happen!
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