Hello,
I am working with recombinant plasmids and E. coli BL21(DE3) cultures for protein overexpression using IPTG or auto-induction medium.
Recently, I have some problems growing E. coli with different recombinant plasmids which is why I cannot see a trend what is going wrong. My vector is peT28a, which is why I am using kanamycin as an antibiotic in plates, liquid cultures, and glycerol stocks.
1) slimy pellet:
After centrifugation, all of my cultures (i.e. different proteins --> different precultures) resulted in very slimy pellets. I was thinking of a phage contamination which leads to cell lysis. I did cyber gold staining of the remaining supernatant but I could not see any phages. So I prepared freshly autoclaved medium and supplements for the medium but I still got slimy pellets. So I inoculated my pre- and main cultures in a different lab and our lab was intensively cleaned with EtOH and 1.2% NaClO. I also prepared a fresh transformation and streaked my glycerol stocks onto new LB-kanamycin plates to make sure there is no contamination. I only pick a single colony from a plate for the culture.
2) stinky, "yellowish" pellet:
Another time, I got "yellowish" cultures which resulted into very stinky pellets after centrifugation and no protein production. I found similar topics facing this problem. People were discussing Pseudomonas fluoresencs, fungi or Staphylococcus aureus. I was also told I could be yeast, but the media was prepared a day before and looked fine.
https://www.researchgate.net/post/Fungal_contamination_of_E_coli_culture
https://www.researchgate.net/post/BL21DE3-PLysS_cells_give_a_slimy_and_sticky_pelletand_have_a_pale_wood_yellow_color
I am thankful for any advice or suggestions! I am really frustrated right now and clueless what to do since I did not have any of this problems in the past.
We had the problem of contamination before (also with pinkish pellets) which is why we cleaned the whole lab but right now I am the only person working with cultures. I change the kanamycin stock 50 mg/ml in MiliQ on a regular basis (~ once a week while it is stored at 4 °C in a fridge)
Normally I prepare precultures from the glycerol stocks. Since the problem occurred I streaked out my stocks and could not see any obvious contamination and I also made a fresh transformation. I used to work with a Bunsen burner but now I switched to an air flow hood and plates.
Streak out your cultures before harvesting. I would look at the MilliQ water, streak it out and make sure it is not loaded with some gram negative organism. Sometimes the water filters can grow bacteria to very high levels. Usually contamination is from the indiviuals aseptic technique not the room you are working in.
I streake out everything and prepared everything new using a different autoclave and syringe 0.2µm filters and new chemicals, antibiotcs, etc.
One of the previous contaminated cultures looked like this:
Any suggestions what this could be?
Nadine Gerlach : i am getting the same contaimination issues in E.coli. Can you please help me in understanding how you resolved this issue.
Thanks
Hey, we solved the problem by discarding all chemicals used for the media and also new antibiotics as well as intensive cleaning of the lab and equipment. In the end all senior scientist I asked thought it was rather a problem with antibiotic that any contamination. I hope you wil solve your problem soon!
- Badges
- Science topic
- Similar topics
- Engineering
- Mechanical Engineering