Hello everyone,
I know that having problems with protein expression is a common problem but when I started the project I didn't have that problem. My protein was expressed, I purified it and everything was fine. Until that one dark day, when I couldn't express it anymore. I didn't do anything differently. I then tried different conditions like shaking speed, different temperatures, different times, I did new transformations, prepared a new LB stock, I tried different flasks and adding glycerol. Nothing works (none of my colleagues has any problems btw). I wonder if somebody else had a similar problem and if you were able to solve it and how.
Next, I'll try TB media, I'll check if I can express another construct and check for mRNA.
Thanks :)
I suppose your biomarker is antibiotic resistance. Whether your organisms are growing on plates with the correct antibiotics or not, I will first check if I still have the correct plasmid construct by doing a plasmid isolation and PCR or restriction digest analysis. This should be your first shot or have you done it already?
Did your expression host cells grow in all of those conditions without expressing your target protein? or Did you observe no cell mass and no protein expression?
If your expression host cells are able to grow in those conditions, the problems will be most probably associated with your expression plasmid. Thus, you need to consider the recommendations given by Valentine. You need to isolate, purify and analyze your plasmid. Check the sequence of your target gene in your expression plasmid.
If your expression cells are not able to grow in those conditions, you need to check quality of your glycerol stocks of your expression cells. How did you make your glycerol stocks? How do you store them? Did you use dry ice to put your glycerol stock prior to innoculation the agar plates? Did you use the right antibiotics with the right concentration in your agar plates as well as your protein expression media?
i strongly suggest you check the plasmid construct again to be sure it is still what you had in the beginning. At least to rule out any doubt about that.
Try different expression method (for example auto induction expression) with the same plasmid.
I would work in parallel.
If the sequencing reveals nothing out of the ordinary, your expression is the problem. You then have two methods to compare.
Hope this helps !
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