For years we have seen variation in RFP produced in E. coli. We have used different promoters and RBSs, but always the same RFP gene (see this as an example: http://parts.igem.org/Part:BBa_J119137).
We decided to grow cells producing GFP vs RFP and save the conditioned media. We used 0.22 micron filter after pelleting cells, and then grew new cells in this conditioned media. The exact same freezer stocks of RFP-encoding cells produce a lot of RFP if they are grown in RFP-conditioned media but do not produce RFP if they are grown in GFP media.
We also found that if we grow RFP-encoding cells with parafilm covering the culture tubes, they do not produce RFP but they will with good aeration. However, the cell density on both cultures (+/- parafilm) is the same.
Any insights or suggestions are welcomed.
Hi,
the fluorophore of GFP needs to undergo ring formation and oxidation. You can actually produce GFP in its pre-fluorescent form when growing E.coli under anaerobic conditions. That possibly explains your parafilm results (in addition to the different amounts of energy the cells get under aerobic vs. anearobic conditions.
I am not sure whether the experiments with conditioned media lead into a useful direction. It could simply be a requirement / consumption of media components.
Cheers Markus
Thanks Markus. You might be right about the parafilm case. The conditioned media experiments have to be something else though.
Markus,
The attached image shows what happened when we grew 3 cell strains with DNA construct built to produce mScarlet. U, C and W each have a different T7 RNA polymerase promoter. Cells were grown in 30 mL with aeration. Next day, media centrifuged and filtered to remove cells. Fresh U, C and W cells grown in all 3 media types (9 tubes total). We also replicated this design but added extra nutrients to each condition (9 more tubes). You can see that all three strains produced mScarlet only when grown in W media which in the original 30 mL culture was the only strain to produce substantial levels of mScarlet.
What were the OD's, viable cell density and RFU of the cultures in the initial round of cultivation? Which strain was used and how was expression induced?
Cheers Markus
All NEB T7 Express cells with 0.1mM IPTG.
ODs were all similar (30 mL first round as well as 3 mL second rounds), about 1.2 OD at 590 nm. The values in the graph show RFP/OD.
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