I tried to check Pb toxicity and accumulation efficiency for my recombinant E. coli strain which contains Pb binding protein in metallothionein ligated in pSB1C3 vector.
I used LB media but I got precipitated Pb in case of with Pb(NO3)2 and PbCl2
Also I tried to use phosphate buffer only but also I got Pb precipitate before starting my experiment.
I used Saline solution (NaCl 1%) to avoid precipitate in case of PbCl2 but I couldn't get good accumulation results.
Hi!
I studied the metal resistance of Cupriavidus metallidurans CH34 and of other bacterial species during my thesis.
I used 284 medium or a modified version of the 284 medium with MOPS and Beta-glycerophosphate instead of Tris. These two media were used to study the metal resistance of Cupriavidus Metallidurans CH34 (Mergeay, 1985). I also used the 869 medium diluted 10x with other bacterial species...
I know that pH can be an issue sometimes ...so, maybe check also the pH...
I hope this will help you!
Cheers,
Steph
Hi
I think you need to check the pH with a buffer so that Pb does not precipitate
hi,
I think you must use free Pb media or you can use any media that you have with blank media (without bacteria) .
Hi,
I think you must use media with low Phosphate because in high phosphate media precipitate Pb .
Best,
Ahmad
Dear Mohamed,
Lead is one of the most difficult metal to investigate in laboratory experiments, because it very easily precipitates or forms complex ions. I conducted a large number of experiments with Pb and observed many unique chemical properties of this metal.
1. Effect of pH
I observed precipitation of Pb when pH of solutions was higher than 6.1-6.3. I observed this process even if I dissolved in the water only salt of Pb (PbCl2 or Pb(NO3)2, without any other chemical compounds. This precipitation is connected with Hydrolysis and can be easily overlooked if concentration of Pb is lower than 0.5-1.0 mM.
Conclusion: If pH of your LB media was above 6.1-6.3. most of Pb could precipitate. If you want to conduct experiments with lead pH of nutrient solutions or any media has to be maximum 5.9-6,0
2. Effect of phosphates
Lead very easy and quickly precipitates with phosphates and this reaction is observed irrespective of pH of solution. The product of this precipitation is not Pb3(PO4)2 but different types of pyromorphites, for example Pb5(PO4)3Cl. All pyromorphites are almost insoluble compounds. The formation of pyromorphites is connected with a large drop of pH of nutrient solution or media, which depends on the concentration of Pb salts (a higher concentration of Pb the higher drop of pH) and buffering capacity of solution. Plant physiologist use complete nutrient solutions to grow plants and for a treatment with Pb (one to several days) phosphates are removed from both control solutions and experimental (with Pb) solutions.
Conclusion: Use of phosphate buffer in experiments with Pb is not acceptable, because even all Pb can be precipitated. However, it could be possible to use the standard media, but for the time of treatment with Pb phosphates have to be removed from the media.
Summing up
If you send me the complete composition of LB media and describe general conditions of your E. coli culture (e.g. pH) I can propose you how to avoid precipitation of Pb in your experiment.
Best regards
Eugeniusz Malkowski
Plant Physiologist
Dear prof. Eugeniusz Małkowski
Thank you very much for your helpful answer, I really was confused.
So, In the fact I tried many experiment using lead salts like Pb(NO3)2 or PbCl2, using LB media or using phosphate buffer solution or using NaCl 1% Solution. Maybe the best results which I got were in case of PbCl2 dissolved in NaCl 1% solution to avoid precipitation of lead, but also in this case I faced to problems; 1st is lower solubility of PbCl2 in cold water, and 2nd is NaCl 1% solution not have any nutrient for bacterial growth.
About your question (LB component): Its composed of Tryptone 10 g, Yeast Extract 5 g, NaCl 10 g, dissolved in 1 liter then we adding 100 ul of NaOH (10 M) to adjust the pH to 7.0.
About my purpose of my work: I make new bacterial strains (which contain Metallothionein and Pb binding protein for accumulating Pb from pollutant circumstance, and I want to check its efficiency compering control one. So I don't matter about kind of media or solutions I just want to expose the two different strains to different concentration of Pb and other heavy metals.
Thank you very much for your kindly help.
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