8 replies to this topic

[Adrian K]

Hi All,

Sorry for the lengthy question. This question had troubled me for quite sometime.

Lately I had transformed one of my recombinant clones (in pet46 vector) into Rosetta-gami™ 2(DE3)pLysS Competent Cells. I found that it had low transformation efficiency , <20cfu per plate [relatively low, as I concurrently transformed with rosetta-gami B(DE3) plyss, and BL21(DE3)], and after I had stocked it in 20% glycerol stock, I find it very hard to revive the recombinant strain, even with and without the presence of antibiotics in the culture media.

Same recombinant vector was transformed in rosetta-gami B(DE3) plyss, BL21 (DE3) and NovaBlue Giga singles, but I got no issues at all. All protocols was standard protocol from manufacturer Merck, fresh newly purchased Carbenicillin, agar LB etc.

I had asked the Merck local technical support, and their reply was:

"pLysS containing hosts express Lysozyme, they are much less stable in glycerol stocks in the freezer after they are transformed. These hosts should be thawed only once and should be used for protein expression after transformation, but not be stored in glycerol stocks for future protein expression."

How true is that?

So my question was:
1) is this also happen to you as well that Rosetta-gami 2(DE3) plyss competent cells is relatively harder to transform?
2) is this also happen to you as well that the recombinant strain are hard to revive from glycerol stock?
3) if I were to store the recombinant strain for future use, what would be the best method?
4) possible for me to manually grow "Rosetta-gami 2(DE3) plyss", and make it competent by my self using Cacl2 method?

Billion Thanks for your answer and sharing.

Adrian

[protolder]

Hola Adrian, I´m continuosly expressing some kinases in rosetta gami DE3 plys and I haven,t any problems.I´m sure that you have a constitutive expression of your protein in the three strains that you use because the repression in them isn´t very effective. Make a master culture with LB and a bit of glucose (0.2 g/l) grow short time untill OD 1-1.5 and concentrate by centrifugation ,if you want, before adding glycerol to frezzing. Of course that you can prepare your own competents bacteria from any strain. In fact, mines, were given for other lab and I make my competents for electroporate. Check phenotypes and add antibiotics at the culture for make comp.in order to mantain episome. Good luck

[Adrian K]

Hi,
Thanks for your reply, Just curious, are you using:
rosetta gami B DE3 plyss
or
rosetta gami 2 DE3 plyss

Thanks

[protolder]

adrian kohsf, on 27 October 2010 - 11:23 PM, said:

Hi,
Thanks for your reply, Just curious, are you using:
rosetta gami B DE3 plyss
or
rosetta gami 2 DE3 plyss

Thanks

Hola again Adrian I´m using rosetta-gami DE3 pLysS nor B nor 2, and checking my expressions there isn´t costitutive expression in lb glucose, because I´m  repressing by glucose and lacIq of episome. In your other cases BL21 and Rosette gami " you haven´t lac Iq and could have constitutive expression. Good luck

[Adrian K]

Hi protolder,
Thanks for your prompt reply but however my problem is not with the expression issue. I have problem with the transformation and glycerol stock revival.

[protolder]

adrian kohsf, on 28 October 2010 - 02:10 AM, said:

Hi protolder,
Thanks for your prompt reply but however my problem is not with the expression issue. I have problem with the transformation and glycerol stock revival.

Yes, I know. the efficiency depends of the lot of competents because the pure plasmid has been obtained in other subcloning strain. if you make and stock with a culture expressing recombinant protein (unless uninduced) , depending of the protein the culture loses viability and frezzing and tawing means a little part of viable bacteria.

Cheers

[ntgxw]

I started to use rosetta-gami B DE3 pLysS competent cells for transformation and further expression. I met the same problem that the revival from glycerol stock failed. I do not know why....

[Adrian K]

ntgxw, on 15 January 2011 - 09:31 PM, said:

I started to use rosetta-gami B DE3 pLysS competent cells for transformation and further expression. I met the same problem that the revival from glycerol stock failed. I do not know why....

can you tell me which batch of RG B(DE3) plyss competent cell you are using? maybe ours were in the same batch.

[ntgxw]

adrian kohsf, on 16 January 2011 - 05:45 AM, said:

ntgxw, on 15 January 2011 - 09:31 PM, said:

I started to use rosetta-gami B DE3 pLysS competent cells for transformation and further expression. I met the same problem that the revival from glycerol stock failed. I do not know why....

can you tell me which batch of RG B(DE3) plyss competent cell you are using? maybe ours were in the same batch.

I tried again by thawing the stock completely rather than inoculating the surface to revive the cells. And it worked. Now I am doing expression assay.