Cloning problem: only frameshift mutants survive - (Aug/13/2007 )
I am trying to subclone an insert to pGBKT7 plasmid. The problem is, after ligation and transformation I get very few colonies (0 to 10, max) and after miniprep and sequencing I always find that all of them have various frameshift mutations in the first exon of the insert or in the plasmid itself.
I would think that the protein product of this insert is toxic to bacteria, thus only frameshift mutants survive. However, it is NOT an expression vector (that is, it can be expressed only in yeast, not bacteria). Has anybody experienced similar problems? What is the explanation for all this?
I would think that the protein product of this insert is toxic to bacteria, thus only frameshift mutants survive. However, it is NOT an expression vector (that is, it can be expressed only in yeast, not bacteria). Has anybody experienced similar problems? What is the explanation for all this?
Honestly, I am not really sure, but do you have a promoter region and a start codon in you insert in your insert dna?
Honestly, I am not really sure, but do you have a promoter region and a start codon in you insert in your insert dna?
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OK, yes, the plasmid has a T7 promoter region (not sure, is it just for sequencing?) and some yeast-specific promoters upstream; also, my insert is a full-length transcript with a start codon. Does that mean it can be expressed in E.coli? If it is so and it is toxic, how do I overcome this problem, what do I do? I am very new with all this, so most basic suggestions will be highly appreciated. Thank you!
It is possible that the yeast-specific promoter is partly functional in E.coli and it is producing enough of your protein to be toxic
Also - a longer shot - are you using the BL21 host strain? If so you may be getting some expression driven by the T7 promoter (assuming it is close enough to the coding sequence) as this strain has T7 polymerase inserted into the chromosome.
Also - a longer shot - are you using the BL21 host strain? If so you may be getting some expression driven by the T7 promoter (assuming it is close enough to the coding sequence) as this strain has T7 polymerase inserted into the chromosome.
I am using DH5alpha strain.
So if there is some expression of my protein in E.coli, what can I do about it? How do I prevent toxicity? Should I try a different host strain? ANy other ideas? Thanks.
Where does the insert come from? I am wondering if there is an error in the amplification primers? AKa did you use PCR to make your insert? And if so, could this be a mistake in the primers? Are all the mistakes in the same place? Sometimes, companies do make mistakes when making their primers.
If the insert comes from another plasmid, might there be a problem with the insert here? Are all the mistakes in the same place?
But assuming it is toxic... well you can change expression plasmid. Something with tighter control. There are plasmids for expressing toxic proteins.
But before going down that road, you could try growing your cells at a low temperature, say 25 celsius. THis is one of the usual methods of expressing toxic or badly expressing proteins. If it is being expressed, lower temperature cultivation will reduced expression rates.
DH5 alpha won't give any T7 expression, so that is not the problem. I assume you are trying to clone the gene using E.coli, then express it in yeast. If that is the case, the toxicity may not be an issue when you take it to the yeast host, so you only need to turn off expression of the protein in E.coli. perneseblue's suggestion of using a lower growth temperature is a good one. What yeast promoter is the insert coupled to? It may be possible to repress it.
If the insert comes from another plasmid, might there be a problem with the insert here? Are all the mistakes in the same place?
But assuming it is toxic... well you can change expression plasmid. Something with tighter control. There are plasmids for expressing toxic proteins.
But before going down that road, you could try growing your cells at a low temperature, say 25 celsius. THis is one of the usual methods of expressing toxic or badly expressing proteins. If it is being expressed, lower temperature cultivation will reduced expression rates.
Thanks for all suggestions, I will try.
The insert comes from another plasmid, pME18S. All frameshift mutations I've seen so far are random, not in the same place, that's why I suspect there should not be anything wrong with the insert...
I am subcloning this insert into pGBKT7 for use in a yeast two-hybrid assay as a bait. Can anybody suggest any other yeast "bait" plasmids, which have may have lower expression levels and would be less toxic for my bugs? Thank you.
you can also add 0.2 % glucose to the plates and liquid medium, this is sometimes done when (willfully) producing toxic proteins in E. coli (Frangioni and Neel, Anal Biochem 1993: 179-187). Another thing that has been described to enable the production of toxic proteins is the presence of 2 % ethanol (in liquid medium) (Donnelly et al., 2001, Protein Expr Purif)
I do not know anything about yeast bait plasmids. But you should try a very low copy vector in order to minimize the toxicity of the plasmid insert.