10:20 A – 12:45 A
Literature
- Xie lab paper on single-molecule imaging of TF binding live
- Bickmore lab paper on NIPBL affects euchromatin compaction in a cohesin independent manner
Ph project
- Project meeting update. Key next steps: transcriptome and compaction (show effect of cluster alterations)
- slides from Ajaz for meeting with Xiaowei
Review writing
- read review, start re-organizing from the top.
New outline
- Pecoud & Ycart the two state promoter
- Lesson: very strong binding + equal output implies koff is small and kon is small ~quadratically increases CoV (approaches ~ 1/koff^2)
- Relation to data: long residence mode is called bursty: highlighted and studied in Raj 2006.
- Sanchez Derivation: General steady state: review Sanchez examples
- repressor looping ? (Also the Vilar and Saiz model?)
- ‘dual activation’?
- PloS CB paper — the average is completion TIME not expression average, KINETIC quantities
- variance in completion time
- Moving forward: Extending Markov models from promoters to enhancers: Current enhancer ‘thermodynamic models’ do not provide estimates of variance.
master equation approaches
- upper bound (sanchez 2011)
- upper bound and iterate (Munsky 08?)
Notes on Sanchez paper
Details of Sanchez method
- collect terms of essentially tri-diagonal process of
Sanchez examples
- simple repressor: ‘Strong operator is more noisy’ for same average expression. (easily visible from noise equation)
- dual repression
- repressor looping
- simple activation
- dual activation
Protein variation (maybe not space to address very much of this)
- Sanchez 2011 protein variation follows mRNA variation just by scaling translation burst size b (in the case where protein decay is slower than mRNA decay, and now protein delta replaces mRNA delta. mRNA delta is now only affecting b).
- protein burst size (beta) is gamma distributed if protein life longer than mRNA life
- Add that if protein lifetime is very short,
- noisy strong operator can be good source for stochastic state switching
Troubleshooting Hybes
- passage Kc167 cells
- 4 new coverslips of Kc167 cells (test Green again, control for embryos)
- cells all washed off before/during fix.
- Try 2. Add cells straight after polylysine, don’t let poly L dry out.
- let attach and settle overnight, fix first thing tomorrow morning.
color analysis
- yellow 06-11 data suffers from substantial uncorrected drift
- first 8 images of 06-24 B suffer from extreme uncorrected drift.
- manual STORMrender through:
- BX-C s4
- BX-C s5
- Y 05-27
- B 05-31
- K 06-01
STORM
- microscope issues: ports scrambled.
- Josh/Hazen fixed ports.
- STORM2 DAPI cube not working at all. Did the 405 bp filter get put back?
- attempting mercury lamp illumination: filter cubes don’t seem to have any excitation filters in. DAPI still doesn’t work.
- checked 2 coverslips of en-embryo sections. each has only 1 mid-stage embryo, very difficult to find.
- old trolox seems to have strong background in red channel. Can’t see en stain (last time only visualized it with TIRF though anyway…)
- O/N STORM of Yellow 100kb locus in Kc cells with 540/560 feducials. lookin’ good.