I'm studying expression of an enzyme in BL21 star cells (cloned in pet 28b) , the protein is expressing well in the soluble fraction and it is active. But the cell mass obtained is very less in the case of LB + IPTG system. As I understand it, autoinduction media can help increase cell densities, but needs some fine tuning? I have a few queries reg autoinduction:
1) Is it good enough to grow the pre-inoculum culture in LB, or should I use a non-inducing (MDG) medium?
2) Should concentration of kanamycin be increased to 100ug/ml?
3) What components of the autoinduction media (like ZYM-5052) have a role in increasing the cell density (I read MgSO4 is one...)
4) Will using autoinduction media be better than using a rich medium (2XYT, TB) + IPTG system?
i can suggest you o use the Enpresso growth media: http://biosilta.com/products-support/enpresso/?doing_wp_cron=1377605177.4447131156921386718750;
If the problem is limited to biomass productiviity but protein expression and solubility are good, it may allow you to mainain the IPTG induction and to obtain biomass/mL about 20 times higher respect the LB.
good luck
manuele
I will suggest to use IPTG or other inducer in the late exponential phase, in my experience it is important not to use rich media, you may find a recipe in a couple of my references. You may also try a pulsed fed-batch, that is simple cam be applied in both shake flasks and bioreactor.
Is your protein toxic to the host cell. That might be the reason for low cell density. Try using the BL21pLysS or similar ones that reduce leaky expression. Also you might want to grow the culrures at 16-18c overnite post induction and reduce IPTG to0.1 to 0.3 mM.
Avinash - read the paper that Gary links to. It is excellent and highly detailed, and will give you all the answers that you are looking for. In summary:
1) LB is fine for the starter culture. As you say, you get active sample from LB+IPTG.
2) Yes. The autoinducing media contain phosphate buffer, and phosphate gives E. coli resistance to kanamycin. At 50 ug/mL, kanamycin is not selective in phosphate, and even at 100 ug/mL it's only bacteristatic rather than killing.
3) All of the components of the ZYM-5052 increase the cell density. That is why they are there. The paper details how each component was optimised.
4) Depends. There are many factors that affect the final level of protein that you get out. The advantages of the AI system are that it is comparatively cheap (especially compared to TB), it is low maintenance (set the cells shaking at 20 and come back in two days. Less work is always good), and you get excellent cell densities. However, some proteins give better yields at lower levels of induction (AI is inducing at very high levels), and as mentioned above, if your protein is slightly toxic to the cells, then you may find that optimising IPTG gives you the best results (if you use Tuner strains of E. coli, then you can optimise IPTG all the way from 1 uM to 1 mM. Sometimes you need _really_ low levels to get the best results) .
I would try everything in small scale first, to optimise the conditions, and then scale up. A couple of days' work to optimise may well save you weeks of repeated preps further down the line.
Gpeople at NIEHS grew up about 2 liter of ecoli, spun to pellet the ecoli, and then used this to inoculate other flasks. I don't know the protocol, but you can contact Gary Powell and Bob Petrovich on Researchgate. It worked great. I hope this helps.
grow your cells on TB broth and u can use 100mM of the sorbitol and glucose solution during the time of inoculation.grow it for more time like 25 degree for 16-18 hrs..
You may have to bubble O2 gas into the culture since O2 tension can become limiting as cells reach higher densities.
- Badges
- Science method
- Similar topics
- Biological Science
- Genetics
- Gene Expression
- Protein Expression