lab meeting outline

try and make new figure showing location among screen hits of the hypoxia hits with oe v 2 on x axis and 2 v 1 on y axis. emphasize that the majority of the transcription factors are not in inducing conditions when screened, whereas the hypoxia genes are.

you should also see where cell cycle genes fall and see if you can add this to the plot.

note: this will require you go through the images and say for sure which ones are expressed.

data on Rox1-YFP in HEM1::LEU2 background. Contrast with previous results. Explain that the major difference between two experiments was 1) plating OD 2) normalized time in imaging media. I conclude that the previously observed noise characteristics may be due to observing Rox1-YFP expression at different stages in progression to heme-starvation.

I also observe that ROX1-YFP is expressed at much lower level in HEM1::LEU2 background than in HEM1+ background and that the range of expression over which ROX1 expression can be controlled is small (~2-fold).

making deoxygenated culture tubes

add 10ml YPD + 2% glucose to vial

add 100ul Resazurin

cap the tubes--make sure to get the petroleum stopper top a bit wet so that when you close the screw top on it, it can rotate and fully tighten down.

bring water bath to rolling boil

place 9 tubes in boiling water for 10m

while tubes are still hot, sparge with N2, while bring tubes down to rt with water bath.

wait until following day to see if any tubes are leaking (based on indicator color change clear-> pink

protocol for ROX1-YFP induction

put 1 matchhead cells from freshly streaked cells into 100ul

put 50ul of this into 10ml deoxygenated media and allow to grow at 30' for ~20h

prepare microscopy plate (treat with conA). place plate on microscope and remove media from target wells. (note: it takes about 5m for the plate to stop moving once you put it on the plate-holder--you should have it stop moving before adding cells, so you can take high quality images immediately after plating.)

take 2ml and spin down in microcentrifuge tube. resuspend in SDComplete.

plate and wait ~1-2m until cells adhere to slide at appropriate density.

begin time-course

rox1 expression coordinated with ribosomal protein genes

McKnight lab has shown that when you grow yeast to saturation, then starve them for > 4h, then allow them to grow in a chemostat (constant glucose supply; dilution rate 0.1 hours ^-1; limited oxygen), they undergo metabolic cycling. HAP1 expression seems to correlate with O2 levels and ROX1 expression seems to peak just after peak of dO2 (dissolved oxygen).

Note that they only see metabolic cycles in two prototrophic strains, and not in lab strains BY and W303.