Hi All

We are currently trying to express a protein using the Origami strain, and we (quite optimistically I admit) tried using our std protocol from our Rosseta strains (2h 37C, induction and transfer to 18C), with the result that we had very small cell pellets. Does any of you have experience with this strain, and if so, which protocols do you use? Can Origami work at 18C at all?

I am looking forward to meet (hopefully) many of you in Paris soon.

Cheers

Bjørn

Hi Bjørn,

you can grow Origami cells in principle at low temperatures like other strains. In your case it could be a protein-specific effect, did you try with a standard protein that should have no issues in any conditions?

The original mutations in the precursor of the Origami strain (trx-, gor-) lead to a significant growth defect which was restored by another mutation as described here: PMID: 11588261. Probably the cells aren't very happy in low temperatures. The yields are generally lower than for other BL21 strains. We don't use the Origami strain very often, only when a user has an established protocol and insists on using it.

Maybe it could help to inoculate with a higher ratio of preculture to final culture. We have sometimes seen (not with Origami but BL21(DE3)RIL cells) that inoculating with 1:50 instead of our standard 1:100 ratio gave a faster growth (obviously) to the OD for induction and higher yield of cells after overnight incubation at 18°C. I don't know if this could be useful for all strains or proteins.

Best regards

Hüseyin

P.S.: I forgot that the main reason for not using Origami is that it is kanamycin-resistant for some stupid reason. For most of our constructs we use kanamycin for selection, so Origami can rarely be used. We haven purchased the Origami2 yet which is not resistant against kan.

Hi all,

we have done extensive benchmarking of the Origami versus BL21 plysS and Shuffle (for the exact strains and results see : Nozach, H et al., High throughput screening identifies disulfide isomerase DsbC as a very efficient partner for recombinant expression of small disulfide-rich proteins in E. coli. Microb Cell Fact 2013, 12, 37) using our standard ZYP5052 37/17 protocol with no major problems of Origammi growths (from the text:  " thanks to the removal of the antibiotics used to maintain the trxB-/gor- genomic mutations in Origami (DE3) pLysS and SHuffle® T7 Express lysY, the growth rate of the three strains were very similar. An increased lag phase was observed with the Origami B (DE3) pLysS and SHuffle® T7 Express lysY but at harvesting time, in stationary phase, the optical density of the 672 cultures was in all cases around 12 (with less than 10% difference)". As stated, the trick is to remove at the protein expression culture step the antibiotics that control the genomic mutations in Origami (DE3) pLysS (very unlikely to be reverted during the growth). We keep  the antibiotics when we prepare the competent cells. As stated by Huseyin, we almost always (not in the study above to be able to compare) double the inoculum to speed up the process (1/20 instead of 1/40).

If you look at the paper you will see that for 28 proteins of the study, Origami/Shuffle was a disaster compared to BL21 (DE3) plysS ("The low number of soluble constructs in Origami B (DE3) pLysS and SHuffle® T7 Express lysY is directly associated with low production levels. In practically all cases these two strains produced lower amounts of soluble fusion protein than BL21 (DE3) pLysS and out of the 28 DRPs,
none would be favorably produced in Origami B (DE3) pLysS or SHuffle® T7 Express lysY as opposed to BL21(DE3) pLysS".

Overall, in our hand the origami has always been a disaster and we used it in probably less than 5 cases in production phase in the last decade... Maybe that is the only way to produce your protein but don't be too optimistic about it. The Shuffle seem to work slighly better in our hand.

Renaud