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Origami B cells grow too slowly!!


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#1 yuyim

yuyim

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Posted 29 January 2004 - 11:33 AM

Hi to everybody,

I am trying to use Origami B (DE3) host cells (from Novagen) to overproduce a GST-fusion protein but I find lots of problems to grow and transform the cells. Even before being transformed, they grow very slowly (two days in the incubator at 37ºC) and the colonies have a very bad appearance. The transformation efficiency is extremely low (following all the manufacturer’s recommendations).
Does somebody know any trick to grow and transform these cells?
Thanks.

Lucía

#2 labsorcerer

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Posted 06 August 2004 - 10:27 AM

Hi Lucia,

I've used electrocompetent cells, and I just grow them in LB. They do grow SLOWER than usual, but they shouldn't take 2 days at 37C to grow. Try this:

1. Subculture 0.2 mL of fresh overnight (ON) into 5 mL LB and use at midlog phase (this is
best for more compromised strains like Origami and will likely give you better
transformation efficiency than using an ON culture which you can use for healthier strains).
2. Harvest cells by dividing the 5 mL into three 1.5mL eppendorf tubes.
3. Pellet at 14,000 rpm for 30sec-1 min. Aspirate supernatant.
4. Add total 1 mL cold autoclaved deionized H2O, vortex and combine into one tube.
5. Pellet at 14,000 rpm for 30sec-1 min. Aspirate supernatant.
6. repeat steps 4-5 (using one tube this time)
7. Add 1 mL cold autoclaved 10% glycerol. Vortex.
8. Spin at 14000rpm for 1min. Aspirate supernatant.
9. Resuspend pellet in 100 uL sterile cold 10%glycerol. Store the cells on ice for use or snap freeze and store in -80C in 40 uL aliquots.

gives you about 3 aliquots. Scale as needed. Transform with your plasmid (try different amounts of plasmid depending on your concentration of DNA).

For your protein expression step:
What may help is streaking FRESH colonies before inoculating your LB+antibiotic (use only the one required to maintain your recombinant plasmid), use a large inoculum from your freezer stock to streak, though. I know that after a month I usually can't get my cells to grow in culture. Also, if you don't need to use Origami for your fusion protein you can always try BL21(DE3) or another faster growing strain, and if solubility is a problem with these you can try growing them at 15C, 25C or 30C (compare on an SDS-PAGE).

Hope this helps.




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