9 replies to this topic

[chrom]

Hi everyone,
I have a gene cloned in pRSETa vector that has an ampicillin resistance selection. I transformed this plasmid in BL21DE3 E.coli expression system and I get no colonies. I tried checking the amp concentration on LB plates, changed the comp cells but the problem never resolved. I simultaneously transformed this clone in rosetta cells and bingo! I get the colonies. Now, the problem is, we know that rosetta cells are derived from BL21 and are meant to overcome codon bias during protein expression. What I dont understand is at the genetic level why is it happening this way? Is toxicity a factor only for BL21 and not for rosetta or DH5 alpha cells?? Ur replies are eagerly awaited.
Thanks:)

[Vini]

Hi

even though u hv written that u changed the comp. cells, are u sure that the new set was good enough....i mean, did u check its transformation efficiency??? And, it don't think that  rosetta cells are free of toxicity probs....

[chrom]

Yes I had set up the control. The transformation worked in BL21, so i m sure the cells have no problem:). Yeah ur right rosetta shouldnt logically resist toxicity, if it exists!

Edited by chrom, 03 December 2009 - 06:03 AM.

[Vini]

chrom, on Dec 3 2009, 08:31 PM, said:

Yes I had set up the control. The transformation worked in BL21, so i m sure the cells have no problem:). Yeah ur right rosetta shouldnt logically resist toxicity, if it exists!

how much amp concentration r u using in ur plates????

[chrom]

100ng/ml working concentration

[Vini]

chrom, on Dec 4 2009, 11:42 AM, said:

100ng/ml working concentration

100 ng/ml???? are u sure???....thats way too less........i use 50 ug/ml working conc......

[HomeBrew]

Too little amp would not stop transformants from arising on the plates...

Since you have checked your competent cells and are sure they're competent, and since your transformation protocol works (or else how would you have checked your competent cells?), this leads me to believe that the problem is not with your recipient cells, but with your clone...

When you transformed your clone into the rosetta cells and got colonies, did you check them to see if they actually had your clone in them?

When you checked your BL21 cells for competency, did you transform them with the pRSETa vector alone?

What are you trying to transform?  Is it a ligation mixture, or plasmid DNA?

[Vini]

HomeBrew, on Dec 5 2009, 09:38 PM, said:

Too little amp would not stop transformants from arising on the plates...

Since you have checked your competent cells and are sure they're competent, and since your transformation protocol works (or else how would you have checked your competent cells?), this leads me to believe that the problem is not with your recipient cells, but with your clone...

When you transformed your clone into the rosetta cells and got colonies, did you check them to see if they actually had your clone in them?

When you checked your BL21 cells for competency, did you transform them with the pRSETa vector alone?

What are you trying to transform?  Is it a ligation mixture, or plasmid DNA?

yeah, that really would not stop the transformants from coming up on the plates........but I was wondering that its way too less to even stop junk colonies from appearing! maybe, as HB pointed out, what u see with rosetta cells, are actually junk colonies and not really ur transformants. that would be worth confirming.....

Edited by HomeBrew, 06 December 2009 - 04:31 AM.

[chrom]

Oops! sorry people...thats not 100ng but 100ug/ml
I have always used this concentration for all transformations so far and never had a problem
Coming to BL21 cells as HB asked...i have transformed some other clone with prseta to check the same but not prseta vector alone and i got good colonies. So i guess BL21 cells are doing fine. I even took cells from my labmates to be sure that my cells arent gone bad and they behaved the same way.
Talking abt rosetta, I am doing protein expression studies, though havnt checked the gene yet. I am actually tranforming a plasmid DNA
Thanks for all ur answers:))

[HomeBrew]

The proper control would be to see if the BL21 cells take up the vector alone by competency transformation.  If they won't, you'll never get your clone in them...