The culture will definitely takes longer time to grow in a fresh broth. Their viable cell count may decrease over a longer storage at 8C and such cells exhibit longer lag period of growth cycle. Use early or mid log phase cells to get reproducible OD600 values!

I agree with Shamsher: the preculture will take more time to wake up if you start from an old plate. This should not affect the production of th recombinant protein unless the clone is unstable e.g. if the protein is toxic and ontrol of expression is leaky. As a matter of precaution, it is better to keep strains as stocks deep frozen at -80 C. Start from a fresh saturated (overnight) culture in rich medium such as 2 YT and make it 20 % glycerol by adding 1/2 vol 60 % sterile glycerol. Store in cryotubes at -80 C. To start a fresh culture, don’t thaw the stock, but scratch the surface of the frozen stock with a sterile toothpick and use it to inoculate the medium.

Hi there Martina,

The optimum OD600 at which to begin induction of protein expression is quite system dependent, and may vary depending upon on a number of factors. In general, however, ~0.6-0.8 is standard. If you ever try auto-induction growth medium I suspect you will find the OD is higher when the bacteria begin expressing your protein.

I strongly recommend fresh plates for expression cultures. I consider plates that are under 2 weeks old (and have been stored at 4C) to be ok, but fresher is absolutely preferable. I dislike the use of glycerol stocks for expression cultures, as the bugs have a tendency to push out the genes/plasmids over time and this can lead to bad cultures and expression problems. Instead I think freshly transformed are better, and I would also make sure that the competent cells you use are aliquoted to minimize the number of times they are thawed (so they remain competent for longer).

If you are seriously concerned about the final OD of your cultures it may be worth checking the OD every 30mins - 1 hour to ensure that it is consistently rising over time and is not the result of the culture dying (which could be a sign of phage or that your protein is toxic). We used to find with some amyloid proteins that the OD would never rise above ~2 even in Terrific broth, which we took to be due to the expressed protein killing the bugs.

Hope this helps!

Best wishes,

James

Unless you calibrate a profile considering OD600 of cell vs their viable count (usually standard plate count) it's difficult to predict accurately the relation between cell number and the OD600 of the E. coli cells. Alternatively, grow and sediment about 500 mg of fresh bacterial cells, prepare cell conc such as 10, 30, 50, 70, 90 and 100 mg/ml wet cell mass and find out their respective OD600 values. This profile shall serve as a calibration curve of cells.