# User:Bohao Liu/Notebook/Research/2013/12/11

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## ARO80 Thoughts and Plans

• T is half period...yes it's weird, but less confusing than using w all the time
• t is the time along the experiment
• I is the average nuclear intensity

### TO DO

• Negative Control (As asked by first referee)
• Same timings as normal experiments (e.g. 3hr, 10min imaging), but no oscillations at all and measure end intensity values : WT,GAP1,WAP1
• Currently I have a WT trial where +tyr starts at T = 0 and then I record everything

### What the first referee wants

• Clearly show the role of permeases
• So we have shown it quite clearly with GAP1, now onto WAP1
• Are they physiologically important
• Not sure how exactly we are addressing this other than looking at the bandwidth

### What the second referee wants

1. I find it very odd that cells do not respond at all at high frequencies.
• From my results, I get a response at high frequencies. e.g. at T = 60, t = 0, I = ~30; at T = 60, t = 18, I = ~50
1. In measuring the frequency response of the cells, the authors acquired data at different rates for different experiments. For instance, when the drive period was 1 min, the acquisition period was 30 sec – but when the drive period was 180 min, the acquisition period was 10 min. Taking two different fluorescent images every thirty seconds puts a lot of stress on the cells. It can also photobleach your reporters. Without the proper controls to make sure these effects are not happening I don’t really trust the data. Plus, if you are only going to report the value of the reporter at some arbitrary time after the beginning of the experiment, why do time-lapse at all?
• The methods we came up with for my set of experiments have consistent acquisition periods and total lengths. The time-lapse is to ensure that the accumulation is running as expected and also to make sure that we did not overshoot the maximal intensity i.e. it never decreases.
1. In section 3.1 and 3.2, the number of sample cells varies greatly in each oscillatory condition, from 7 to 111 cells. This can significantly affect the variation of protein accumulation. The number of quantified cells, especially T = 6 min, should be made comparable to each other. In general, this makes me wonder what is really going on in these experiments that the number of cells varies so greatly.
• While I don't have exactly consistent number of cells between different frequencies, I do have at least 300 in each so I think this addresses the concern
1. The authors did not mention whether the experiment in Section 3.1 and 3.2 were replicated. If not, additional experiments are needed for better quantification.
• No replicates currently....
1. Figures 6 and 7 are very confusing, and the discussion of them is extremely hard to follow. It’s not clear to me what these figures add to the study.

Plus, are there any error bars for those plots?

• In progress, looking at this stuff now
1. The ploidy and mating type of the strains used in several experiments is not consistent. For example, in Section 3.5, the control and Gap1p strain is Mat-a, whereas the Wap1p strain is Mat-alpha. Also, while most strains used in the experiments are haploid, the experiment of overexpression of ARO80 used a diploid strain (Fig. S1 legend) - this increaset of gene copy number in the pathway can affect the pathway induction. These points need to be clarified.
• The strains I have been using are all consistent: yMM1164(WT), yMM1238(Gap1 - del), and yMM1239(Wap1 - del) are all Mat a HAP1+ARO9-yECitrine-KanMX.