110316 lab mtg

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Identification of HetR targets during heterocyst development:

One of these things it not like the other… Identification of HetR targets during heterocyst development Britt Flaherty Lab Meeting 3/14/2011

HetR is the master regulator of heterocyst development:

HetR is the master regulator of heterocyst development HetR was originally identified as a MCH phenotype when overexpressed ( Buikema and Haselkorn , 1991). HetR mutants do not differentiate heterocysts after nitrogen step-down ( Risser and Callahan, 2007)

HetR is the master regulator of heterocyst development:

HetR is the master regulator of heterocyst development HetR is expressed in response to nitrogen deprivation ( immunoblot – Zhou et al., 1998) Time ( h ) of nitrogen deprivation HetR binds DNA in vivo (Zhao et al., 2004)

HetR is the master regulator of heterocyst development:

HetR is the master regulator of heterocyst development Kim et al., in press

ChIP of HetR will identify its DNA targets:

ChIP of HetR will identify its DNA targets

FLAG tagged HetR complements a hetR∆ phenotype:

FLAG tagged HetR complements a hetR∆ phenotype Strain: WT Plasmid: none Media: BG11 N- Strain: ΔhetR Plasmid: hetR:FLAG Media: BG11 N+

The FLAG tag is inconsistent on a western blot:

The FLAG tag is inconsistent on a western blot One crummy western gave signal with a FLAG antibody… but never again.

ChIP with primary anti-HetR:

ChIP with primary anti-HetR Step 1 = design primers with a similar Ct and one product = no template controls

ChIP with primary anti-HetR:

ChIP with primary anti-HetR Step 1 = design primers with a similar Ct and one product

ChIP with primary anti-HetR:

ChIP with primary anti-HetR Step 1 = design primers with a similar Ct and one product

Slide 11:

Step 2 = optimize fixation and look for enrichment ∆Ct = Ct gene of interest – Ct rpoB ∆∆Ct = ∆ Ct gene of interest in WT- ∆ Ct gene of interest in deletion strain Fold Enrichment = 2 -∆∆Ct ChIP with primary anti-HetR

Slide 12:

Step 2 = optimize fixation and look for enrichment ChIP with primary anti-HetR

Slide 13:

Step 2 = optimize fixation and look for enrichment ChIP with primary anti-HetR

ChIP with anti His and anti FLAG:

ChIP with anti His and anti FLAG

ChIP with anti His and anti FLAG:

ChIP with anti His and anti FLAG

Next Steps:

Next Steps ChIP with anti-His with a normalization control (HetR on a plasmid without the tag). Use new primers against the hepA promoter. ChIP my newly-made HetRFLAG mutants. Begin integrating HetR:his into the chromosome. Publish many many papers and graduate.

Slide 17:

Jim Golden Susan Golden Anabaena Neil Blake Tong Dawn Anna Anthony Yong Ick Spencer Arnaud Julie John Susan C. Funding CMG Training Grant Committee Eric Allen Steve Mayfield David Ryan Raul