How might you break CCS to determine what is necessary for CCS’s function? List the residue (amino acid) and it’s location (or what you would break), the reason you chose the location. Design primers that you would use to for site-directed mutagenesis to mutate that residue.

Molecular Biology lab

Testing the phenotypes

Your goal is to test your strains that you made in the yeast transformation lab. You will do this by comparing growth of a wild-type strain, a strain missing Sod1 (sod1∆), and a strain missing CCS (ccs∆). (SOD1 = gene; sod1∆ = gene has been knocked out or removed from the genome; Sod1 or Sod1p = protein)

Yeast lacking Sod1 activity, whether it is because the Sod1 protein itself is gone or because the CCS is in not functioning (or gone), cannot grow on media lacking lysine. This is because superoxide (O2-), which is an ROS (reactive oxygen species) is thought to attack one of the proteins in the lysine biosynthetic pathway. Specifically, superoxide will attack proteins with Fe-S clusters (think a cube with Fe and S on alternating corners). This damaged cluster falls apart and the protein in the pathway no longer functions which means the yeast can’t make their own lysine and need to get it from their media. This growth assay is a very quick and easy biochemical/genetic test to determine if there is Sod1 activity. Yeast are about 90% reliant on CCS to activate Sod1. So if CCS is not there or broken, Sod1 can’t get activated and there will be no growth on SD-Lys media.

Obtain a SDC (synthetic media complete – with all – amino acids) plate and a SD-Lys (synthetic media but missing only the amino acid lysine). Divide the plates into 4 quadrants and label them with the strains listed and with a name for your strain. You will take either a sterile wooden stick or flame sterilized inoculating loop and fill the quadrant with the yeast being very careful to not “color” on the lines. This should look like you made very tight zig-zags that almost overlap, but not quite overlap (see the picture). The goal is to have the most yeast at the outer edge of the plate and the least yeast at the inner corner. This will help you see if a strain grows well, OK, not so well, or not at all. Both plates should have the identical strains. The SDC plate is the control (you should see growth for the strains). The SD-Lys plate is the treatment. Grow the plate at 30˚C for 2-3 days.

Assess the growth and take a picture of your plates. Include this in your lab notebook and your paper.

Questions

⦁ What was the result of the experiment? Include a picture and a discussion of what you saw.

⦁ How might you break CCS to determine what is necessary for CCS’s function? List the residue (amino acid) and it’s location (or what you would break), the reason you chose the location. Design primers that you would use to for site-directed mutagenesis to mutate that residue.

Last Completed Projects