lola chat

stumpf lab work

Transcript

1. Eliseo Papa Data munging for the LoLa project
2. Nitrogen stress response in e.coli
3. That was way too complicated! • Well studied pathway, suited to study stress response • What does the bug do under N limitation? • We are interested in two TFs: NtrC(glnG) and Nac • genes regulated by Nac are transcribed via σ70 • Ntr is σ54 dependent • Nac transcription is activated by NtrC
4. More on ntrC and nac • To the last count nac regulated 25 genes / 10 transcription units • ntrC thought to regulate up to 2% of genome • under N limiting conditions: ↑ N assimilation and tries to keep the bacteria growing • Part of glnALG operon (also glutamine synthetase, NtrB sensor kinase) • ntrC (and nac) bind to an inverted repeat sequence which can be far from TSS
5. Experimental setup time N spike t0 tOD t3 t9 N limited • Can bacteria sense N limitation and prepare the Ntr response? • What happens when it’s shocked back to N abundance? • “inverse” response? or does a different initial state matter ?
6. Chip-Seq analysis • Tells us where does the Tf protein binds on the genome • correlates with regulation
7. ChipSeq analysis 1. peak calling - Find the peaks 2. motif analysis - Does the binding sequence make sense? 3. annotation - Where do the peaks lie? Which genes could they be regulating?
8. Peak calling • SISSR, MACS, FindPeaks, BayesPeak, ... 28 different ones.
9. Motif • up to now ntrC consensus was: tGCaCCaaaatgGTGCa
10. Annotation • not so trivial in bacteria • ntrC can act from far (but we don’t know just how far) • lots of overlapping genes
11. Tiling array • Genome level RNA levels • 60bp resolution
12. Annotation/2 • we can be more intelligent in calling genes regulated by ntrC and nac
13. Integrating it all
14. glnALG operon
15. We need more (for modelling) • ntrC exerts its effect when phosporilated • Can we use a time series of Tf binding data and transcriptomics data to obtain a proxy of Ntr(P) ?
16. Thanks for the hospitality! and special thanks to Angelique & Maxime