Hi Avishek,
Could you be a bit more specific about what you need to do.
As nifL is the negative regulator in nif operon, I want a in-frame mutation in nifL gene so that it will produce a truncated protein.
So you want to introduce early stop codon in the plasmid? Probably a tandem PCR might be tbe easiest. Do you have a full map of your plasmid? I can take a look.
no, dats the problem, I dont have the full map of that plasmid. It contains 5 plasmids. While curing that plasmid, that bacteria stops growing.
Sorry for the way I'm asking, just trying to understand your system. So you have 5 different plasmids coexisting there or a big multicistronic plasmid expressing 5 different ORFs? If all plasmids are coexisting together would be difficult to isolate the one you need to work on it.
Did you try to extract the plasmidic DNA (by mini prep for instance) and run it in a soft agarose gel (0.5%) to see whether you might size-separate the different plasmids?
If plasmids can be separated you can design primers to sequence it (also will help to know the promotor/terminator pair driving its expression).
Once you have the plasmid and primers the mutations to create the truncate mutant are easy to perform, then you can transform all the plasmids (4+mutated) back in your receptor system.
Fell free to ask anything! I have 5 different plasmids coexisting together. I have already extracted the plasmidi DNA and run on soft agarose gel. their I am getting 5 separate bands. And another question appear on my mind, when I am trying to remove the palsmids, cells are not growing. How can I transform the mutated plasmid in receptor system (wild having all the plasmids) system?
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