- This topic has 5 replies, 4 voices, and was last updated 2 days, 4 hours ago by Opher Gileadi.
January 13, 2022 at 16:45 #4160
Some of my best friends are using freshly-transformed BL21(DE3) cells to inoculate directly overnight liquid cultures for expression, without selecting on agar plates. It is against my instincts… but am I wrong? what do people think?
January 13, 2022 at 16:55 #4161
it works great – we do it all the time.
You can recover after transformation into a starter culture (which you can archive as a glycerol stock) and use to inoculate expression cultures, or you can recover after transformation in autoinduction media. In both cases, we leave out the antibiotic during recovery, then add antibiotic for overnight incubation. We’ve done side-by-side comparisons of these two approaches vs. the traditional method of selecting a single colony from an agar plate, and there is no difference in the final protein yield.
January 13, 2022 at 16:55 #4162
I was using such an approach but not to produce recombinant protein but to induce its activity in vivo (in bacterial cells). It requires robust antibiotic selection (so kanamycin is Ok but I would not use ampicilin selection). And I do not see any advantage of not using plate selection of extransformants prior to protein production although I am rarely using single colony inoculation ( usually I am using several to avoid any bias)
January 13, 2022 at 16:59 #4163
good point about antibiotic choice Krzysztof! we’ve only tested this using kanamycin selection
January 14, 2022 at 10:48 #4164
Hi all, Opher it has been a while since we met 🙂
Chris answer has been our default protocol for almost 2 decades (2003). It works for almost everything whatever the antibiotic(s) in the soup, Ampicilin included. On our default protocol, at the end of the transformation half goes for preculture and half on agar plates. The precultures are used for HTP screening, glycerol stocks or even for flasks inoculation (in that case we put the 50 ul in 5 ml LB o/n as the starter). The plates are there to be kept 1-2 weeks in the case that we need to redo a bigger cultures. Glycerol stocks are there if we need to scale up later on. You can have all the details in my papers (where I describe how we do 1152 transformations/precultures/agar plates in parralel). You can start by our Jove video (in open access) and the paper that comes with it : “The next day, the preculture is used to inoculate the test expression and for preparation of glycerol stocks. Put the agar plates in a refrigerator, as a back up. If necessary, starting from the agar plates or the glycerol stocks, the test expression could be re-done by inoculating a fresh LB preculture directly”.
When starting a culture from a plate I always scrap many colonies as I have seen in the past with colored clones that once in a while one colony on the plate is not colored; taking several colonies decrease the bad luck to pick a wrong clone in the middle of a plate.
We have seen few cases where starting from a glycerol stock/liquid transformation/old plates was not giving at all the same yields or poor reproductibility with the previous batch. This is the case for complexes or using strains with chaperones or proteins with multiple SS bridge… After a first batch that doesn’t look good we then redo a fresh transformation each time, this is not always solving the issue…
We has seen several times, for tricky proteins that even starting from the SAME culture of 350 ml that we split for the 3 scale routine purification of the team (325 ml on AKTA, 24 ml on our 24dw and 2 ml on our DW96 Tecan protocols) that even if everything is stricky identical, we sometimes get better protein quality/quantity from one of the DW purification compared to AKTA, or vice versa. This is mostly due to local protein concentration/contact time differences during the binding to the Nickel in batch versus column, but not only… Despite what some of the PI thinks protein production/reproductibility is not alway easy to domesticate :-).
January 20, 2022 at 20:53 #4165
Thanks all for very useful responses! I was specifically worried about ampicillin but I’ll follow your expert advice (Renaud).
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