I want to express a fungal gene under the control of it's own promoter but there is no reliable online tools to find out where exactly the promoter of that gene is, how many bases upstream or downstream? Anybody has any idea how to do this? Although I searched a few online tools to find the promoter but mostly the online tools are specific for a particular organism.
Thanks in advance
Hi Latif,
Which organism you want to express this gene in with its own promoter? Do you know (or been published) that whether this promoter works in that organism?
Thank you Yau,
I am working on Verticillium dahliae, I have constructed gene deletion strain and now I want to construct complementary strain, for that I want to express the gene by introducing it under it's own promoter but I don't have any idea how far is its promoter, upstream or downstream etc..
Latif,
I work on Arabidopsis, for the plants we usually consider 2kb upstream on the gene as the promoter when we want to make such a construct. I'm not sure whether it works the same for your Verticillium or not, but I suggest you to refer to some of the published works in your field and double check how far in the upstream region of the gene they consider and take as the promoter.
If there is no published data of this gene out there at all, you can just clone different lengths of the promoter region (ex. 2 kb, 2.5 kb, 3 kb..etc) and fuse it to a GUS gene for expression test. It will take longer time, but a systematic approach is more scientific than randomly pick a length. Online tools can give you a prediction, but no warranty.
Below is a link to a published paper with similar topic. Their material is fungal maize pathogen Cochliobolus heterostrophus, and yours is fungal Verticillium dahlia. Their constructs were tested using transient gene expression assay and stable gene expression assay.
http://www.ncbi.nlm.nih.gov/pubmed/8232214
Paper title: "Transient and stable gene expression in the fungal maize pathogen Cochliobolus heterostrophus after transformation with the beta-glucuronidase (GUS) gene."
AOA Latif,
There are many online tools which can predict the promotor of any gene in any organisim, (the sofware is not sepecific for specific organisim). You can select 3ooo upstream sequence as cited by many papers. i.e., you can search www.fruitfly.com. hope this will helps
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