I think there is likely to always be significant variation, especially between different conditions. One possibility is to have prolonged incubation times so that the growth disparities becomes insignificant, try to let the co-cultures incubate much longer than what you are using (if you stop at 3 days, try 5 or 6 days instead) and see if this reduces the variability.

Michael J. Benedik Thank you!

I leave these co-cultures for 14 days post inoculation with B, production typically starts on ~day 8 post inoculation with B.

So there will be day 0 inoculation with A and B up to day 14, establishment for 1 day to 14 days of A before inoculation with B which is left for another 14 days. eg, 14 day establishment of A before addition of B for 14 days will be 28 day experiment.

Across these producing days, the concentrations produced vary significantly across triplicate experiments.

Is this just because of there being 2 bacteria, that there are too many variables to control? Or is there a way to control this and increase repeatability?

Thank you so much for your response

One thing to look at is viable cell count of both strains after your incubation period to see if something is happening to one of the strains. I would expect there to always be some variability but probably not extreme.

Do you know enough about the synthetic pathways to consider whether there might be another approach to what you are trying to do? For example if B is produced a precursor that A needs, can you grow up a culture of B, remove the cells then culture A in the conditioned medium that carries the precursor?

Michael J. Benedik

thank you!

I have tried plating every day post inoculation to identify whether one strain is taking over the other, and toward the producing days, A is the only visible strain in the culture, whether it has antibiotic production or not, which is very strange.

There is not a lot known about this strain, I am sending it for transcriptomics soon to try elucidate the pathways.

It is good to know I am going along the right lines as I have tried culturing A in B supernatant as well as autoclaved B with no production detected.

However, I did add media constituents to these, I think it may be worth not adding those componets to see if B is utilising part of the media more than A, which is inducing the expression.

I am currently trying another media called “streptomyces antibiotic media“ to hopefully remove the need for the coculture.

Very interesting. This raises the question of whether strain B is really needed for production by making some co-factor, or is just an inducing agent that turns on production of A.

Have you tried the opposite of your regime, grow the B culture first and then add A to it. My thinking is that if A is killing B you may not be getting enough growth of B. So perhaps growing B to saturation for maximal cell number, or maybe just as a mid log phase culture. That way you will always have plenty of cells in case they act as an inducer of some type.

Michael J. Benedik thats what I am also thinking, whether the actual physical contact is required.

That is a very good idea, thank you! I will try that.

I think I will try culturing A with and without B cells, so try growing in supernatant to see if is is a nutrient depletion caused by the rapid growth of B compared to A, rather than it’s physical presence. As well as culturing A in different days of B growth, like you suggested.

As for the variation, I am not really sure what to make of this. I am hoping the new media will induce stable expression so I can state in my thesis that the variation is definitely from the 2 bacteria and not just not repeatable from A.

I really appreciate your help!