I wish to identify and clone TA pair from bacterium which genome was not sequenced, how I should start...
I guess your toxin/antitoxin are proteins? Can you isolate them? If yes, you can make an Edman degradation, which gives you at least a part of the amino acid sequence. If it is possible to do mkae a complete degradation, you´ll just need to do a back-translation, and you´ll have the gene. If you get only a part of the sequence, you can search for sequence homologies, e.g. blast against ncbi. Maybe you´ll find sequences of other organisms, which are similar to yours.
Once you have the sequence, you can order the genes and do with them what ever you want.
Thank you Uwe, yes in my case these are proteins and your suggestion is very helpful for me, however if I am not able to isolate them, there is any other alternate method as it is not easy to identify target protein complex (TA) from thousands of proteins of a bacterial cell.
Since you can't isolate the protein of interest to go back from the AA to DNA sequence, you could construct a genomic library of your unknown bacterium and insert fragments of the genome into vectors to be expressed. If your gene pair codes for a specific phenotype, you could select for that colony based on phenotype and then sequence the vector to get your gene pair of interest. Note that this is tedious work and would only work if your gene pair encodes for an identifiable phenotype.
With the decreasing cost of whole genome sequencing, I would personally suggest that you extract the genomic DNA of your bacterium and send for whole genome sequencing. Downstream bioinformatic analyses have come a long way and would annotate your gene pair of interest as well as the rest of your unknown bacterial sample
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Methods
- Cloning