I've been trying to detect nitrite from bacterial cells cultures in which the bacteria had a construct expressing bNOS. Tried with a commercial kit (from sigma. This one also converts nitrate to nitrite, so I could measure both) but it failed miserably (lots of negative values for my samples. The standards were ok). Thought it was my construct, so I tried again with a different one. Nothing. Then simply tried measuring nitrite with the home made Griess reagents (which I know works because it's what I used to determine if macrophage are activated, and it works perfectly there). Though that maybe the levels were too low, so changed to using the DAN reagent (fluorometric). Measured my unknown samples (different dilution) with exactly the same gain as for the standard but, again, more negative values, including for my positive control (MRSA which has a bNOS and should be producing NO, which will be converted to nitrate and nitrite). I did this with 3 different constructs, but every time I get negative concentrations of nitrite. I've though about the LB media interfering with the assay, but I make my standards with same volume of media I'll be using in the assay, and the standard works. Also possible that none of the constructs is actually functional, even though at least one I know for sure it is expressing and the gene was synthesised (with codon optimisation), which will be incredibly bad luck. Anyone had a similar problem, or know what could be going wrong? Any help/ideas would be appreciated.
Thank you
Hello, Claudia.
Would you check whether the transformed bacteria have plasmid vector bNOS along with WT bacteria and a positive, transformed E.coli with any plasmid vector available? You should see different plasmid vectors in your bNOS-bacteria and transformed bacteria with other vector groups, but not in WT bacteria. You should have enough, replicated plasmids in both gel bands.
I think you've done nearly everything for the measurement of NOS activities. Have a look at the upstream. See whether transformation, replication and activation of your plasmid gene in the transformed bacteria are properly done.
Best wishes.
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