We are going to make a construct with T5 promoter. We are going to order M15 strain from Qiagen. However, I could not find the product except one PDF file about it. Do they stop selling M15 strains? Do anyone know where are the other resources to get the strain?
Dear Liang
i'm not an expert of T5 promoter because generally i'm prefer T7, pCold or pBad but to my knowledge the T5 promoter do not require any specific E.coli strain so in theory you can use also the standard BL21(DE3).
However if your plasmid do not codify for the lacI, to have an better repression before IPTG induction you can choose a strains with lacIq (e.g https://www.neb.com/products/c3037-neb-express-iq-competent-e-coli-high-efficiency)
good luck
manuele
Have you tried contacting one of their sales reps?
The probably do not sell M15 alone, but included in one of their pQE kits. Beware of getting M15 from other sources, as Qiagen's version comes with a plasmid overexpressing the lac repressor (without which apparently you tend to select for T5 promoter deletions, see PMID 19171063)..
Hope it helps!
Thank you guys so much for your answers! They are super helpful! I think we will try to use JM109 strain which has the lacIq. It may be a little leaky, I will let you know how it goes. Maybe I will try C3037I from NEB, too. I talked to Qiagen represent they did not tell me about the kit. They suggested JM 109 and XL1 blue. Now I am searching for it, seems like they used to have M15 in a kit but not now. The one they are selling is the one with C-terminus vector set with 3 vectors but no cells. It is also $635, a little pricey. The C30371 was suggested by NEB representative. I will do a little research on it. Yes we love T7 in our lab too! We have BL21(DE3) cells too. We are trying to find cells can work with T5 is because we want to use a vector with his tag on C term, nothing on N term, which is a little tricky to find. So we found pQE 60, which is kind of ideal for us. That is why I am trying to figure out the best cells for the vector. :)
Ahh ok!
However pet21 and pet24, which are standard commercial vectors have His on C-term.
And moreover using the PIPE cloning approach (see attached file), using just 2 primers you can easily add a his at the C-term of your gene if you have it cloned in a plasmid for the expression of the un-tagged version.
That is sooo helpful!! Thank you so much Manuele!!! How do you know so much about this?
Update of my question just in case there are people with similar problems: We actually used JM109 DE3 which is not ideal, because it has T7 promoter. Our construct is T5 promoter. So I gave it a try without adding IPTG. I grew the transformed bacteria in 2L LB for 6 hours and I got enough protein. It seems to be the right size on the gel. Probably because T5 is a strong promoter and my protein is not toxic to the E. coli. I got very lucky. Now we are moving forward to the next steps.
Hello everybody. We are trying to use the pQE80-L vector and apparently, the M15 pREP4 strain is not available. Does anyone have any suggestions on what can be used? Has anyone had success using JM109?
Thanks.
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