I write here to seek help on isolating a lipase gene from a bacterium LTR48. I am trying to amplify lipase from a bacterium which is most likely a Pseudomonas baetica (dendrogram attached). But NCBI database has very limited sequence data available for this organism (few rpoB, 16SrRNA gene and ITS sequences available). Moreover the organism's whole genome has not been sequenced yet. So, my filtered search for lipase in NCBI refseq nucleotide or protein or gene database yields me a sets of lipases (which are either triacylglycerol, esterase, lactonizing lipase, secretory lipase etc.) from different Pseudomonas species such as aerugenosa, putida etc. I tried to download few of the protein sequences and design degenerate primer using Genefisher2 (http://bibiserv.techfak.uni-bielefeld.de/genefisher2/submission.html). But I couldn't come to any result.
How do I go about designing primers for the lipase from this bacterium which is likely a Pseudomonas baetica but whose sequence information is not available? How can I design primer for each of the different types of lipase? The bacterium shows good lipolytic activity on specific media. What are the various steps that are generally undertaken in designing primers of this kind which are genus targeted.
I would be very much grateful if you can help me out on this.
i need to have primers to amplify the lipase gene from my bacterium which is likely a P. baetica showing potential lipolytic activity. Universal primers not available as such.
You have a few options, I presume your goal is to clone the lipase gene. 1) You could purify the lipase and do an N-terminal sequence of it and use that to decide which lipase you have and generate primers. 2) make a genomic library of your bug and just screen for clones expressing lipase activity. 3) There are many different lipase's in Pseudomonads, so you will never get a universal lipase primer pair. However you can categorize most of the lipases into a few groups and you could make universal primers for each of the groups separately.
Thank you Michael. That helps. Actually i have taken up the task of generating genomic library and protein purification side by side. But wondered if anyone could come up with the idea of designing universal primer.. I believe i will adopt your 3rd point and design number of primers targetted for different proteins.. Thank you.
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