Hi everyone,
I work with Pseudomonas fluorescens EtHAn strain and I 'm facing this issue:
Transformed EtHAn cells grow normally in solid media (LBA + 3 selective antibiotics), but when I transfer the cultures in liquid media (LB + the same 3 selective antibiotics) they don't grow at all.
I tried to transfer the cultures from the solid media in LB + only 2 of the selective antibiotics but they still did not grow.
Noteworthy, this hardship does not apply for all the cultures being tested.
One type of liquid cultures, coming from EtHAn cells transformed with one specific gene (unrelated to antibiotics resistance), grew just fine in both cases.
Any ideas of why is this happening or possible alternative options?
Many thanks in advance.
Regards!
1) Do you have positive and negative Ctrs? If yes, what did you use there and what is the outcome?
2) What is the concentration of your antibiotic? Try maybe different concentrations and different culture volumes!
Good Luck!
What is the mode of antibiotic resistance and/or what antibiotics are use using? If it's an efflux pump, then there should be no issue with liquid vs. solid. If you are using ampicillin and the bla gene, the bacteria secrete the beta-lactamase enzyme that will degrade the ampicillin in a liquid culture and remove the selection pressure. This means your bacteria will drop their amp resistance plasmid.
You may have a selective loss of resistance with 3 antibiotics. Try a single colony from a solid medium and then go to liquid culture. The bla gene for Amp is notorious for artifacts as Katie has suggested.
I've seen a similar phenomenon with E. coli and sulfonamide. The cells formed colonies, but could not be subcultured in the presence of sulfonamide. In this case, what was probably happening is that the cells had sufficient reserves of folate (sulfonamide inhibits folate synthesis) to grow to colonies, but by then there reserves were exhausted. The colonies had a characteristic 'crepe paper' appearance, in contrast to the truly sulfonamide resistant colonies, which were quite dense.
So, I'd take a closer look at the colonies; maybe look at the cells under a microscope too.
i had the same problem before, you have to check the flasks if it clean enough or not as some time the flasks contain some impurities which affect bacterial growth
also you have to make expression and solubility tests in small scale first and run on SDS PAGE gel and see the results if everything is okay you can try different inductions such as Autoinduction and IPTG induction if its okay you can try in large scale
in addition you can use different media such as 2yt for example as some times specific LB media is not suitable enough for bacterial growth.
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