Experimental Biology Experiment Log

Black = No Adenine

Blue = No Leucine

From left to right: 2B RD (AatII), 2B PCR, 2A RD (AatII), 2A PCR, 1B RD (AatII), 1B PCR.

1A accidentally was thrown all over the benchtop.

FUS was amplified out of the vectors at the correct size (1.7kb) via PCR, and the bands look correct for the RD as well, with one at 7.3kb and the other at 5 kb. 

There is a small window when you can see no stress vs stress, and then when the stress condition goes away. 

Stress granules can only be seen at 60x with oil immersion, as otherwise it is difficult to tell whether spots are actually aggregates or something else.

Next time:

NON-STRESS SITUATION: Get to oil immersion and fluorescence within 10 minutes in the non-stress situation, mid-log phase.

KCl STRESS: When you start the microscopy for non-stress, add 1M KCl to the stress condition so that you can get back to it within 15-20 minutes, make a new slide, then you have 10 minutes to get to oil immersion at 60x and fluorescence and see clear stress granules.

Added 1 M KCl to the control (just Pabp-RFP Yeast with Leu pREP1 Empty Vector) and 1 M KCl to FUS/Pabp-RFP Yeast, in addition to having yeast without the hyperosmotic stress.

Will let the stress occur for 30 minutes then go visualize under a microscope. 

All the Ade strains still need time, although there are tiny colonies on the Leu EV + Ade EV

ATF1 and CSX1 by themselves.

ATF1 is a similar toxicity to FUS (slightly less toxic, maybe), CSX1 is greatly more toxic. Gene is expressed on the plate on the right.

Sweden Yeast!

Since last posting:

Transformed CSX1, UPF1, and ATF1 into fission yeast and assessed their toxicity. UPF1 is nontoxic, while ATF1 is as toxic as FUS, and CSX1 is slightly more toxic.

Tested UPF1′s rescue ability for FUS (both on leu- vectors, however extracted plasmids from a single colony and was able to amplify both UPF1 and FUS from it, suggesting both are there). UPF1 can suppress FUS toxicity.

Transformed Ade empty vector into yeast, those transformations require up to 8 days to see colonies, but appear to grow well (just slowly)

Transformed CSX1, UPF1, and ATF1 (leucine marker) into  yeast with Ade marker to assess how to select for that and everything.

Tried to do a western for GFP-FUS, however we had no DCIP/NBT so no bands could be seen.

Digested and ligated FUS into pREP1-Ade vector. 

Obtained endogenous RFP-tagged Panb fission yeast strain from Sweden. Panb is a stress granule marker. 

Transformed FUS and pREP1 empty vector (both leucine) into Sweden fission yeast, then will do a spotting assay and check fluorescence under a microscope to see if FUS induces stress granule formation in fission yeast (add-on request from committee meeting)

Extracted plasmids from yeast that had pREP1-FUS already in it and was transformed with pREP1-UPF1. A PCR was done and then run on a gel in the following order: FUS Primers + Yeast Plasmid Extraction, 1kb DNA Ladder, UPF1 Primers + Yeast Plasmid Extraction.

It looks like both FUS and UPF1 are there, which supports what the plates look like as just FUS had a bunch of tiny colonies (along with the CSX1-FUS and ATF1-FUS), however UPF1 had much larger colonies, that when inoculated in liquid medium overnight can grow to an OD of 1.3 (while the CSX1, ATF1, and FUS alone didn’t grow to an OD of anymore than 0.02). FUS should have a band around 1.7kb and UPF1 should have a band around 2.8 kb. Both appear to, although FUS had a nonspecific band at 1kb, potentially due to a poorly optimized PCR. 

UPF1 appears to have been transformed and suppresses FUS cytotoxicity. A rough spotting assay was done earlier today and a more official one will be done in the near future to assess how well it can suppress toxicity.

Transforming pREP1-Ade EV, CSX1, UPF1, and ATF1 into fission yeast.

Performing protein extraction of Budding Yeast with 1x integrated FUS, pREP1-FUS, pREP41-FUS, pREP81-FUS, and GFP-FUS for running a Western Blot later this week. I normalized the OD to 2.3 for everything.