First questions first

The other day we sat down together to take our first joint look at the grant proposal I'm writing. The proposal is still rather inchoate (first time I've used THAT word), especially the experiments section, but it's coming along.

Our model for how the secondary structure of sxy mRNA regulates its expression proposes that the speed of progress of RNA polymerase along the gene determines whether the secondary structure forms before ribosomes can bind and start translating. So I was proposing to directly test whether RNA polymerase pauses or stalls, especially when nucleotides are limited. But one of the grad students pointed out that we first need to show that the secondary structure does indeed block translation. Now she's given me information about a kit that should let us do just that, using cloned wildtype and mutant sxy sequences she's already made.

The kit uses the E. coli translation machinery to translate mRNAs that it makes from a user-provided DNA template with a T7 polymerase promoter. Her sxy clones have this T7 promoter - she's used it to synthesize the RNAs she's used for the RNase digestion analyses. We can use the sxy-1 and sxy-7 mutant RNAs to see if minor changes to the secondary structure change the ability of the E. coli ribosomes to start translation, and we can create a version that lacks most or all of the secondary structure as a positive control, to show how much translation happens in the absence of secondary structure.

It would be scientifically better to do this analysis with H. influenzae translation machinery (rather than E. coli), but we'd need to develop the system from scratch rather than using a kit, and I don't want us to invest that much work into this question. Once we know what happens with the E. coli kit, we can maybe try it with a H. influenzae cell-free lysate.

Too many questions

I just numbered the questions my draft grant proposal proposes to answer, and found that I have 19 questions! This is far too many, unless I'm asking for a zillion dollars, and I know I don't have the skills to administer that big a project.

But some of the questions are much more important and much more substantial than others. So I'll turn some of them into 'subsidiary questions' and "if time permits" questions. I would rather not simply leave them out, because then the grant panel will just complain "Why isn't she proposing to do X?".

Starting when the job's half done

I did a lot of work on the CIHR grant proposal in July, because I was aiming for a Sept. 15 deadline, until I realized we could easily afford to defer it to the Feb. 28 deadline. This makes the job much less daunting.

So I spent part of yesterday reading over what I'd written last summer, and some of what I wrote for the previous proposal (2001). A big part of the work will be organizing what we already know into a coherent story.

It's scarily easy (for me) to forget results of past research. For example, in previous posts here I've speculated that the purine repressor PurR might repress the rec-2 gene. But in the 2001 proposal I wrote that the previous grad student who had knocked out the purR gene had shown that it does exactly that! Now I need to go through his old notebook to find the data - if it's solid (his data usually was) I can include it in the new proposal or even in the purine regulation paper we will be writing.

Next day: The student was a careful worker, but this particular result wasn't very reproducible. That's probably why I forgot about it.