Heterologous gene expression in yeast - a bioinformatician needs your help cloni - (Sep/12/2012 )
I have done mostly bioinformatics for the last 6 years or so and I've been given the task of complementing yeast mutants with three specific mycorrhizal genes. I think I've finally worked out the correct ORFs and can design primers to target the start and end of the protein starting from the cDNA. I even have the yeast mutant on hand and was given plasmids with the corresponding yeast genes cloned in as well as empty vector to do my own cloning.
My stunningly stupid questions:
the vector pRS412 only seems to have a lac promoter with lac Z after the MCS. Will i be able to induce with IPTG in yeast? Will expression of lacZ be at all useful in yeast or is it just for blue/white screening in e coli for the cloning process? I've attached the vector image to help.
I also want to add a 6x his tag and/or GFP. What would be the most efficient way to link the GFP after my proposed ORF sequence?
My yeast mutant only grows in sorbitol, does this make the traditional ways of introducing the plasmid- by transformation at all tricky? Could someone recommend a useful protocol?
I would appreciate any advice or suggestions you might have...
I've figure out that I must introduce a new yeast promoter and wondered if these was any suggestions for optimal placement of the promoter or if I could just stick it in any compatible place in the MCS?