On Yeast And ALS (Part 3)
- 3 minsThis is the third post on my ALS yeast series. The first post describes existing yeast ALS models and the second post describes in more detail the project I am working on this summer: chemical modifier screens in yeast models of ALS.
Current state of the project
We got e. coli containing plasmids with WT TDP43, WT FUS and TDP43 M337V from the Gitler lab. We performed a miniprep using Zymo’s kit and then did a transformation using standard methods (exact protocol here). Two days later we got transformants, which you can see below
The next thing we did was a liquid growth assay (we decided to do liquid assay instead of a spotting assay because that is how our screens will be performed). For that we picked a colony for each plasmid and inoculated them in 3 ml of raffinose. They grew at 30C while shaking at 230 rpm. After the first day we picked a new set of colonies and inoculated them in 3 ml of glucose and they were grown overnight in the aforementioned conditions. We washed the glucose cultures with nuclease free water twice. Then we diluted the cells grown in raffinose to 1:100 for FUS and 1:10 for the two TDP43 strains and 1:500 for the cells grown in glucose, into either glucose or galactose. A picture of the assay is below. The cloudy wells are wells that contain glucose (and are cloudy because of the growth) and the transparent wells contain gal
We were able to replicate the results, which you can see below (note: the order of the wells was slightly different as I accidentally switched some wells around the first time)
The next thing we did was to grow cultures in glucose, wash them and then re suspend them in galactose to make sure that the gene was being expressed. Since the protein is tagged to YFP we were able to see it under the microscope. Below is an image after the cells were in glucose for 2.5h. It is still a really early time point (as aggregation is usually quantified after 6h)
And we took images again after 23h (unfortunately not at timepoints in between). Here you can see that the expression is starting to diffuse again, though more cells are expressing the gene
Finally, given the data above, we felt confident enough in our model to start a screen. We set it up to be able to test different compounds (roughly 3,000) in the 3 different models using two independent transformants to make sure the screen results are reproducible. We dispensed compounds using the Echo (thanks to Alec Ludin for doing it!), then prepared the following dilutions (both in galactose and glucose) TDP43 transformants 1 and 2, TDP43 M337V transformants 1 and 2 -> 1:500, FUS transformant 1 -> 1: 250, FUS transformant 2 -> 3:500 and dispensed 50 microliters into each well. The first 2 rows of every 384 well plate are negative controls (galactose with DMSO) and the last 2 rows are positive controls (glucose with DMSO). We’re still waiting on the results - stay tuned!
Jamie Cayley
Grad Student & TA at Missouri State University