I tried different culture medium for cell induction, including LB broth, pure water, PBS, LB supplemented with PEG polymer, LB supplemented with alginate polymer. In all conditions, cells are alive which has been examined by live/dead stain. But cells cannot be induced in LB supplemented with PEG polymer. I am curious why that happens? I use the AHL as the inducer. Cells are engineered to produce GFP when AHL is present. I also tried other genetic engeered cells and corresponding inducers including cells containg IPTG inducible gfp.
Appreciate your suggestions and help!
If the heterologous system is dependent of AHL, then the density of cells play a big role in addition to the culture media conditions (they could be expressed better at higher cell densities). Another common condition that improves the expression from natural genome-hosted alternative sigma factors induced, is the growth in culture media with lower concentration of nutrients (i.e. LB 1/10). Slower growth curves, but more chances to have expression of heterologous proteins under a transformed promoter.
Thanks Howard! The cells I used has simple AHL inducible gfp gene. They don‘t have quorum sensing effect. Similar as the IPTG inducible gfp gene.
The nutrient effect also bothers me. I tested the cell induction in PEG+pure water, and PEG+LB. Neither of them shows fluorescence... but for other chemicals like nanoclay, adding LB will lead to much significant GFP than cells w/o nutrient. I will test the condition with LB 0.1X.
Thank you again for your reply!
I used E.coli BL21 p LYS strain for one of my experiment. Induction was given by IPTG. Firstly I tried at 24 degrees but met with failure. Later following few suggestions I used 10-degree celsius and it worked. Hope it helps. Thank you.
NOTE- I expressed a recombinant protein. The expression was good and I could purify the protein and use it for further analysis.
Problem solved. Thank you guys! Seems the PEG (Mn=360), HEMA monomer and some other small molecules are toxic to the bacteria cells...
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