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HELP!Mini preps resulting in low yields where previously high. - (Nov/08/2010 )

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adrian kohsf on Thu Nov 11 13:34:15 2010 said:


perneseblue on Thu Nov 11 04:24:04 2010 said:


adrian kohsf on Wed Nov 10 16:59:56 2010 said:


But then, seriously, is phage contamination frequently happen? How do we know when is phage contamination and how we can prove it? This sounds scary...


Phage infestation are rare (unless somebody in the lab/corridor actually working with phage).The symptoms of an infestation is typically goes something like "the culture was growing fine, when it just stopped growing and started being less cloudy/going clear.."

Usually culture failure are caused by
-leaky expression of some toxic gene.
-glassware contaminated by detergent.
-contamination by competitive/predatorial bacteria.
-phage infestation.


My newly purchased ROSETTA-GAMI 2(DE3)pLysS COMPETENT CELLS was growing fine, but it does relatively less cloudy when it grows on plate (compare with nova blue strains and BL21(DE3) strains. The transformation efficiency is also relatively low, and the culture revival success rate is also very low. Do you think is possible of phage infestation?

Thanks.


Do you mean to say that a culture of ROSETTA-GAMI 2(DE3)pLysS is less cloudy that BL21(DE3) strains? There was no sudden drop of OD during normal un-induced growth. If so, I would tend to rule out phage infestation.

-perneseblue-

perneseblue on Fri Nov 12 05:39:30 2010 said:


adrian kohsf on Thu Nov 11 13:34:15 2010 said:


perneseblue on Thu Nov 11 04:24:04 2010 said:


adrian kohsf on Wed Nov 10 16:59:56 2010 said:


But then, seriously, is phage contamination frequently happen? How do we know when is phage contamination and how we can prove it? This sounds scary...


Phage infestation are rare (unless somebody in the lab/corridor actually working with phage).The symptoms of an infestation is typically goes something like "the culture was growing fine, when it just stopped growing and started being less cloudy/going clear.."

Usually culture failure are caused by
-leaky expression of some toxic gene.
-glassware contaminated by detergent.
-contamination by competitive/predatorial bacteria.
-phage infestation.


My newly purchased ROSETTA-GAMI 2(DE3)pLysS COMPETENT CELLS was growing fine, but it does relatively less cloudy when it grows on plate (compare with nova blue strains and BL21(DE3) strains. The transformation efficiency is also relatively low, and the culture revival success rate is also very low. Do you think is possible of phage infestation?

Thanks.


Do you mean to say that a culture of ROSETTA-GAMI 2(DE3)pLysS is less cloudy that BL21(DE3) strains? There was no sudden drop of OD during normal un-induced growth. If so, I would tend to rule out phage infestation.


Ok, here are the details:

1) I tried to subculture ROSETTA-GAMI 2(DE3)pLysS, BL21(DE3), BL21(DE3) pLysS etc on LB agar plate. I found that it is less cloudy, less creamy and is more watery like with and it does looks semi-transparent unlike the others.

2) I use the competent cells (prepared by manufacturer, follow manufacturer's protocols) and transformed my plasmid (Same plasmid same batch preparation into multiple host) into all the mentioned host strains. It seems that ROSETTA-GAMI 2(DE3)pLysS strains gave the least cfu per plate (~10 cfu per plate for RG2 strains, but the others gave more than ~30 cfu)

3) I do a stock culture for all: the RG2 gave the least recovery from stock culture.

So, how do you think?
Anyway thanks a million for reply me.
^_^

-adrian kohsf-

hmmm.... I don't have anything.

but i am going to make a guess...there might be leaky induction of T7 RNA polymerase, perhaps from contaminating lactose in the growth media. I am assuming that the plasmid used in the transformation expresses a gene.

Try adding 0.05% w/v of sterile glucose (final concentration) to the growth medium immediately before starting the bacteria culture. See if that helps any.

-perneseblue-

Hi perneseblue,
is it recommended if I adding up to 1% w/v sterile glucose into my LB medium? the higher / lower the better?

I will try to do it as soon as possible...
Thanks again

-adrian kohsf-

That might help. The idea is that by adding glucose, you partially inhibit expression of the Lac operon (via cap/crp) which should help in eliminating expression of the T7 promoter. Do you really know that your cells are not healthy? B strains look different from K strains, and you might have perfectly healthy cells. Have you tried induction to see if your protein is expressed?

-phage434-

is it recommended if I adding up to 1% w/v sterile glucose into my LB medium? the higher / lower the better?


Would it be recommended to use 1% w/v glucose as primary energy/carbon source in your media?

That is a difficult question, it depends very much on the characteristics of the protein that is being expressed. Higher cell densities does not mean more functionally expressed protein and not all proteins in insoluble aggregates can be refolded into a functional form. The micro environment changes in a high density medium, affecting intercellular environment which in turn affect protein folding. The only thing I can suggest is give it a try and find which medium is most useful to obtain functional protein. Also note some proteins can be sensitive to the conditions that they are expressed in, and will change if the culture volume is scaled up.

You might also want to explore Superbroth, Terrific broth, 2xYT as example of richer mediums.

As for using higher concentrations of glucose, it would only be beneficial if you have equipment to provide high rates of oxygenation such as air pumps, oxygen enriched air. If not, the inability to meet the oxygen demands of culture will lead to carbon flux bypass into acetate formation. Acetate acidifies the medium inhibiting growth. Acetate also inhibits biosynthesis of some amino acids.

Instead of using glucose as a carbon/energy source, I would recommend using glycerol (1-3%) instead. Glycerol is transported into the cell at a slower rate, so the oxygen demand is not that high.

-perneseblue-

phage434 on Sun Nov 14 13:26:09 2010 said:


That might help. The idea is that by adding glucose, you partially inhibit expression of the Lac operon (via cap/crp) which should help in eliminating expression of the T7 promoter. Do you really know that your cells are not healthy? B strains look different from K strains, and you might have perfectly healthy cells. Have you tried induction to see if your protein is expressed?


Hi phage434, thanks for the info. I had to to regrow my RG2 plyss construct from stock into broth but it failed (yes, it could be due to lots of reasons) and only the second attempt I managed to get it. However, I have yet to induce this strain. But then, the weird thing is other strains such as BL21(DE3) with same vector do not suffer the same faith. I will try to verify again the cfu by using the provided test plasmid.

perneseblue on Mon Nov 15 05:49:50 2010 said:


is it recommended if I adding up to 1% w/v sterile glucose into my LB medium? the higher / lower the better?


Would it be recommended to use 1% w/v glucose as primary energy/carbon source in your media?

That is a difficult question, it depends very much on the characteristics of the protein that is being expressed. Higher cell densities does not mean more functionally expressed protein and not all proteins in insoluble aggregates can be refolded into a functional form. The micro environment changes in a high density medium, affecting intercellular environment which in turn affect protein folding. The only thing I can suggest is give it a try and find which medium is most useful to obtain functional protein. Also note some proteins can be sensitive to the conditions that they are expressed in, and will change if the culture volume is scaled up.

You might also want to explore Superbroth, Terrific broth, 2xYT as example of richer mediums.

As for using higher concentrations of glucose, it would only be beneficial if you have equipment to provide high rates of oxygenation such as air pumps, oxygen enriched air. If not, the inability to meet the oxygen demands of culture will lead to carbon flux bypass into acetate formation. Acetate acidifies the medium inhibiting growth. Acetate also inhibits biosynthesis of some amino acids.

Instead of using glucose as a carbon/energy source, I would recommend using glycerol (1-3%) instead. Glycerol is transported into the cell at a slower rate, so the oxygen demand is not that high.


Hi perneseblue, thanks for your reply. I will definitely look into those media as well as addition of glycerol. Million Thanks again.

-adrian kohsf-
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