This paper
Danquah, M. K. & Forde, G. M. Growth Medium Selection and Its Economic Impact on Plasmid DNA Production. Journal of Bioscience and Bioengineering 104, 490–497 (2007).
Makes the claim that adding extra NH4, Mg, Ca, Glucose and Phosphorus to LB medium will increase the production of pdna. I am currently growing some e coli to test this myself. Here is an interesting article and comments regarding the limitations of classic LB broth.
https://schaechter.asmblog.org/schaechter/2009/11/the-limitations-of-lb-medium.html
Any experts here have insight on this topic?
thank you!
- Amy J.
it is interesting to me how carbohydrate poor LB actually is, ie the e coli are metabolizing amino acids for their primary energy source. Apparently in the early years of molbio, LB had glucose in it but the practice stopped. Would be interesting to know why.
EFFECTS OF SUGAR ADDITION IN LURIA BERTANI (LB) MEDIA ON ESCHERICHIA COLI O157:H7 Article Effects of sugar addition in Luria Bertani (LB) media on
Article Escherichia coli Physiology in Luria-Bertani Broth
glucose in LB tends to lower the pH, and LB is already close to 9.0 where the growth of e coli will cease
Amy, in general most bacterial media used for protein induction is largely composed of something like this:
-1-2% Tryptone, 0.5-1% Yeast Extract, and after that you get a variable amount of salts, buffers, or micro nutrients that are personalized towards your strain and protein of interest.
-Sugar in bacterial media is usually reserved for something like a seed culture or a post-transformation rescue media, like SOC or BHI, where rapid/exponential growth is wanted.
-Remember, constant division is not necessarily a good a thing for heterologous expression of a protein, cells have to juggle resources between division and protein expression.
I'd recommend trying something like 2xYT instead of LB. It works just as well in small as larger ferm scale.
Also if you are just referring to the the TOTAL amount of actual plasmid copies floating around. That is entirely dependent on which E.Coli oriU your plasmid utilizes. They can vary from 5-700 per cell.
https://blog.addgene.org/plasmid-101-origin-of-replication
I have also read that supplying chloramphenicol in the final hours of growth stops nuclear DNA replication but does not stop plasmid replication, leading to a boost in plasmid numbers. I am working with high copy ori, generally
I do appear to be getting more pdna by supplementing LB with additional nutrients, my yield has increased approx. 30% over previous extractions. I add 0.5 grams of a mixture of NH4 sulfate 20%, CaCl 5%, Mg sulfate 5%, Potassium Phosphate 5%, glucose 65% to 5.0 grams LB powder all to 250 ml H20. Growth is faster in this medium, even with 0.015 g kanamyicin added.
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