Dear all
We have prepared metagenomic DNA library and now we are screening this library following the high throughput screening method and using fluorogenic substrates to find Hydrolytic enzymes. As the screening Data is very big so we are looking for the best method to analyse this data to identify the hit.
Any information please!
Hi Theo
Thank you very much for your reply, it is exactly what you mentioned until the use of fluorescence intensity threshold which I do not know it exactly because I am looking for novel enzyme and I am not sure if I can use the value of other known enzyme or no.
I also get results on "Relative Fluorescence Unit", I found that I need to normalize my Data first, and here I am not sure what method should I follow.
>" I am looking for novel enzyme and I am not sure if I can use the value of other known enzyme or no."
You have to use some positive control. You can normalize to positive control (it can be a clone with heterologously expressed known hydrolase in the same screening experiment. Then you can compare both Fluorescence Units: all measurement effects are the same ). Second possibility: normalize to negative control: clone with empty vector. All data with >= 2 times of negative control are you candidates for novel hydrolytic enzymes. And should be screened more times, for reproducibility. You can visualize your data with heatmap in R.
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