14 replies to this topic

[otinanai]

Hello,
I have problem expressing my protein in minimal media. I have successfuly expressed it in LB but in the stadard M9 salt minimal media I cant. Please any help/idea is welcome. I have tried, 3hours induction(IPTG) at 37oC and overnight at 25oC but with no success.

ideas pleaseeeeeeeeeeee

P.S. host cells : BL21GoldDE3pLySs
plasmid : pet16b
tag : 6His
target protein : ~14kDa , soluble

[shimshady]

Are you labelling the protein for NMR? What protein are you expressing, i think i can help if this is the case.

[otinanai]

yes i want the protein for NMR. But now no I didnt label it, I just want it from minimal media for a 1D NMR first and then I will go for labeling with C and N. The protein is a U-Box protein, a part of it. tell me please what do you think, I listen!!!

[shimshady]

Is it actually an expression problem, or is it a cell growth problem. I have always had problems getting cells to actually grow in minimal media, so i have had to use an alternate method for this part. How exactly are you growing your cells in the minimal media? Have you ever take a proton-NMR of a protein, its a giant mess.

[shimshady]

Is it actually an expression problem, or is it a cell growth problem. I have always had problems getting cells to actually grow in minimal media, so i have had to use an alternate method for this part. How exactly are you growing your cells in the minimal media? Have you ever take a proton-NMR of a protein, its a giant mess.

Just to make sure are you using glucose for source of carbon and ammonium (cl or sulfate) as source of nitrogen? Youve made M9 minimal salts correct? Youve used the Vitamin mix and possibly trace metals?

[otinanai]

I'm prety sure that its expression problem and not growth! They grow as fast as they do in normal LB media, so I bet its expression issue there. I know proton-NMR is mess, and nothing special after all, but we just a first blink before we move to expensive labeled media. If we went directly to labeling we had the expression problem again plus the cost of that media!

I use Glucose for carbon source and ammonium SULFATE for nitrogen source. I dont think(and hope ) that there is any problem with sulfate, (is it?)! Yes as much as I concern M9 salts are correct. I also add trace metals BUT there is an issue with vitamin mix. By that I mean I only had Biotin . So Vitamin mix = just biotin... Ι found through net that there shouldnt be any problem with that. Vitamins(espessialy Thiamine) were essential in the past, modern host cells do not neccesarily require these extra vitamins.

I think I gave everything you asked , now what do you suggest?

[shimshady]

It looks like you are doing the right things, the only thing i could think of is that you werent giving the cells the "stuff" to express the protein, but it seems you are. My only suggestion would be to try the vitamin mix (100x). I had come across that same net stuff claiming that the cells only need certain vitamins but i just went with whats been working which is the vitamin mix. If you interested i come across a paper which has helped me express every labeled protein ive used, when i had a cell growth issue, it might work for you.

http://www.springerlink.com/content/g02744...97fa7c&pi=8

Good luck, ill help if i can.

[otinanai]

Thank you very much...
That paper does not really solve "expression problem", more like cell growing problems... But anyway I can only thank you. I will read it thourougly tomorrow!

From a fast blink I was shocked with 8gr/L Glucose !!(from paper)
I thought 4gr/L was the maxium...I'll try that, but also I will try to find some more vitamins (thiamine at least).

[shimshady]

The paper says its trying those cells growth conditions with different amounts of glucose to test glucose starvation and overconsumption. However it says that 4g/L is the stll the way to go. The paper does say that "longer growth rates can result in reduced expression due to cytotoxic effects associated with the plasmid and plasmid gene products exerting selective pressure." It may or may not be an issue for you, but it doesnt hurt to try. Good luck.

[Luria Bertani]

shimshady, on Apr 7 2009, 10:39 PM, said:

The paper says its trying those cells growth conditions with different amounts of glucose to test glucose starvation and overconsumption. However it says that 4g/L is the stll the way to go. The paper does say that "longer growth rates can result in reduced expression due to cytotoxic effects associated with the plasmid and plasmid gene products exerting selective pressure." It may or may not be an issue for you, but it doesnt hurt to try. Good luck.

Hi there, I am having exactly the same problem. It is quite frustrating indeed!

I've made everything a second time- the vitamins, kanamycin, re-transformed my plasmid into BL21(DE3) E.Coli, and made the trace elements fresh. The cells grow quite fine on Agar plates (with Kanamycin) and in LB with kanamycin (50ug/mL). But when they hit the M9 Minimal Media- they refuse to grow! It worked so well last time, I'm just not sure why it's not working this time around. To rule out the plasmid, I've use glycerol stocks from a previous experiment where I know my protein expresses...

The protocol I am following is from http://www.springerl...74871554844506/ [DOI: 10.1007/978-1-60327-058-8]

In a 50 mL M9 Minimal Media stock, there is 100 mg labelled glucose (e.g. 2g/L) and 50mg of labelled NH4Cl (e.g. 1g/L).
Usually I transfer 20uL of cells from freshly saturated LB Broth to 2mL of M9 Minimal Media to 'adapt'.
The only thing I can think of is, perhaps the pH is killing the cells? Phage infection? Something else?

[Luria Bertani]

I've done some more work on this problem- and now my expression works!

It was the trace elements mix (1000x)! The mix tends to oxidise, which makes some metals fall out of solution and become unavailable for uptake by the bacteria. I made up the mix again- and my expression in M9 works. The trace elements are important for the TCA cycle and other metal dependant cell processes. Thank heavens.

Trace elements mix should NOT be dark brown or rusty colour, but a golden/dark yellow colour. Don't keep the mix stored for too long.

Microbiology- part science, part witchcraft!

Cheers
LB

[ILoveJuice]

Luria Bertani, on 10 June 2009 - 03:19 PM, said:

I've done some more work on this problem- and now my expression works!

It was the trace elements mix (1000x)! The mix tends to oxidise, which makes some metals fall out of solution and become unavailable for uptake by the bacteria. I made up the mix again- and my expression in M9 works. The trace elements are important for the TCA cycle and other metal dependant cell processes. Thank heavens.

Trace elements mix should NOT be dark brown or rusty colour, but a golden/dark yellow colour. Don't keep the mix stored for too long.

Microbiology- part science, part witchcraft!

Cheers
LB

Hi!
after I sterile filter my 1000x trace metal, the color is dark yellow too! I'm trying to figure out which metal fall out of my solution, because the concentration might be off after some metals crash out.
my 1000x trace metal recipe is:
a. 50mM FeSo4.7H2O (no problem dissolving it)
b. 10mM MnCl2.4H2O (no problem dissolving it)
c. 2mM CoCl2.6H2O (no problem dissolving it)
d. 10mM ZnSo4.7H2O (no problem dissolving it)
e. 2mM NiSO4.6H2O (no problem dissolving it)
f. 2mM (NH4)6Mo7O27.4H2O (no problem dissolving it) but color turned from yellow to RED PURPLE!!!
g. 20mM CaCl2 (no problem dissolving it)
h. 2mM CuCl2 (no problem dissolving it)
i. 2mM H3BO3 (slight issue with solubility, can see cloudiness) maybe is h or g, not sure

any pointers on solubility issues and oxidizing problems would be greatly appreciated!
THANKS

Edited by ILoveJuice, 31 March 2011 - 02:21 PM.

[mdfenko]

adjust the pH (to 7?)

[ILoveJuice]

mdfenko, on 01 April 2011 - 10:48 AM, said:

adjust the pH (to 7?)

Hi,
Thanks for replying. I don't think it's pH problem, dilute HCl actually helps to keep Iron in solution.

I'm thinking about removing the (NH4)6Mo7O27.4H2O from my trace metal, the dark color might be metal oxides. Any ideas? I really don't want formation of metal oxides.

Thanks in advance!
ILJ

[pDNA]

what IPTG concentration do you use for induction?

Regards,
p