It's very unlikely the construct is toxic to DH5alpha. My guess is either the ligation isn't working or the transformation isn't working. Some general suggestions:

1) If you're using chemically competent E. coli, switch to electroporation.

2) Extremely unlikely, but maybe your 5'UTR has a region that's similar enough to somewhere in pRS316 to allow recombination to excise a big chunk of the plasmid including some of the backbone (your gene doesn't flank URA3 or CEN6 does it?). You should be able to check for vector-insert homology in whatever sequence analysis software you use. If there is significant homology, try a rec- E. coli strain (e.g. Stbl2).

3) Are you PCR amplifying the insert and using this in ligations? If so, you need extra nucleotides on the 5' end of the primer or the restriction enzymes can't cut the PCR product (and there's no easy way to test if the PCR product is fully digested). Make sure there are enough nucleotides in your primers (each enzyme requires different lengths of flanking DNA to work, NEB website provides this kind of information).

4) If you're doing an undirected cloning into blunt ends, you must use a proofreading polymerase for the PCR (Taq leaves A overhangs). Also, you can't use de-phosphorylated vector (to prevent recircularization) unless you specifically ordered PCR primers with 5' phosphates.

I've encountered so much variability in cloning PCR products by ligation, that I don't do it anymore. I always TopoTA (or TopoBlunt) clone PCR products now. It's an extra step because you then have to cut the insert out of the PCR vector, but it ALWAYS works and is worth the extra effort.

5) Are the restriction cut sites in pRS316 the same for the construct without the promoter? If not, that doesn't really control for anything. You need to know if the vector has been properly linearized. Again, there's no easy way to test this (unless you're cutting the vector with a single RE). If 1) - 3) are not applicable, I would suggest redoing the ligations with freshly cut pRS316 (use new enzymes if you haven't recently tested their activity in the same conditions).

If none of these suggestions are helpful, we could trouble-shoot better with more specific information about the procedure (which enzymes, etc.). Good luck!

Dear Mohd,

most yeast promoters work also in E. coli. For example URA3 promoter and TRP1 promoter work in E. coli since you can transform specific strains which are auxothroph for the orthologous genes (for example MC1066) with plasmids containing URA3 or TRP1 with their promoter and the transformant E. coli strains can grow on bacterial synthetic medium without uracil or tryptophan. So if the promoter of your gene works in E. coli and the product is toxic, you will not obtain correct transformants. I know at least two yeast genes which are toxic under their promoter in E. coli (of course it can depend also on the E.coli strain), one of which is DPN9, I don't remember the other one.

In this case, we performed an abundant ligation (at least 30 ul) and transformed directly the yeast with the Gietz protocol that you can find at http://home.cc.umanitoba.ca/~gietz/method.html (it is fundamental you use high molecular weight ssDNA such as Type III from Salmon Testes, Sigma D1626 which increases the tranformation efficiency 100-1000 times compared to  low molecular weight powder DNA that most labs use). Normally I have 100-300 transformants. If your strain transformed with the correct plasmid has a different phenotype from the strain transformed with the empty plasmid check for that phenotype to discriminate cells transformed with the full or with the empty plasmid. Otherwise you should perform PCR, or yeast colony PCR, with external primers. If then you have to sequence your insert, you should perform a PCR  and sequence the amplicon, since there is no chance to obtain the plasmid in E. coli.

Best,

Enrico

I am facing the exact same problem with one of my yeast genes. Plus, we have overexpressed the gene in bacteria and purified it before so there is no issue of toxicity. I did not have any significant homology based on BLAST and DOTTUP results, still I had recently even tried the stbl2 electro-competent cells for the same and got colonies , I did a single digestion and got a shift of about 3 Kb which would indicate the insert but there was no insert release on double digestion and no PCR positive. Currently I am planning to do a ligase-free cloning based on recA independent homologous recombination in DH5 alpha. Any insight would be helpful here.

Hi Aekagra,

I was not able to clone my gene of interest in bacteria, instead I used gap repair cloning in yeast.