donny

I'm working on Saccharmoyces cerevisiae for the first time and am intending to co-express 2 proteins from one plasmid. I have initially thought I can simply clone the genes serially and they will express under the same promoter, like in E. coli. Would this work in S. cerevisiae? I'm asking because I've read many papers and they all use pESC vector such that each gene is under the control of their own promoter. Is this essential?

I have been trying to grow E. coli DH1 transformed with various plasmids in "plain" M9 medium, ie. this recipe with thiamine and NO other supplements like casamino acid, iron, trace metals etc. I've adapted the cells by subcultivation 3x but the growth rate is still significantly slower than rich media like LB. Adding casamino acid definitely increased growth rate but I don't want to use it because I want a real defined media. Are there other ways to increase the growth rate? Would adding iron and/or trace metals help?

I am trying to make DH1 chemical competent cells using the transformation buffer found here. However, my cells look very flaky and clumpy when I try to resuspend them in the transformation buffer. I'm not sure if it's the buffer, the nature of DH1 or something that I've done wrongly. I streaked DH1 cells on LB-agar with 15ug/mL nalidixic acid, then inoculated a colony into 10mL LB with 15ug/mL nalidixic acid for overnight growth at 37C. 1 mL of the overnight cells was then added to 400mL of LB and grown to OD600 0.4. The cells were contrifuged at 4000g for 3min in 50mL falcon tubes. This was when I added the transformation buffer to the pellet and had difficulty resuspending the cells. I tried TSS buffer and got the same results.

Has anyone have such experience too?

EDIT: Just to add that I just took the clumpy part and diluted with water. They seem to resuspend very well. What's going on?!?!