Hi Tom-
Thank you for your feedback on the requested strains and materials. The idea of deleting HEM1 from DFS160 is a great one since, if this initial experiment proves successful, that strain will be generally useful in rewriting other parts of the mito genome with the HEM1 reporter. And as for working with the hem1 deletion strain, I'm hopeful it will be relatively healthy if grown on d-ALA containing plates since the hem1::KanMX deletion I have from Fred's lab (in an S228c strain background) grows essentially normally on YPD with d-ALA.

So my revised plan would be to
1. make a hem1::KanMX nuclear deletion in DFS160 and grow on plates with d-ALA.
2. tranform the strain with pRS415 and the HEM1-HAmito marker with COX3 flanking sequences. Select for Leu+ nuclear transformants on plates with d-ALA.
3. mate the Leu+ transformants to the intronless, Seraphin strain CK520 and screen for respiration defective, d-ALA independent, G418R growth.

How does this plan sound to you? I think it would require only two strains from your lab (DFS160 and CK520), although I wonder if it might also be useful to have the cox3::ARG8m strain for comparison.

My one last question is about the plasmid used to transform the rho0 mitochondria. Will any kind of plasmid work or are there particular origins of replication I will need or need to avoid?

Again, thanks for emailing me today. It sounds like, even in the "lazy days of summer," you're very busy.
With appreciation,
Natalie