Transforming rosetta+chaperone cells with topo plasmid - (Jul/18/2010 )
Hi there
I am trying to produce an enzyme from Carsonella that I have cloned into a topo151 vector.I have opted to use rosetta and chaperone cells to transform my vector into as standard BL21 did not work, perhaps for codon optimization reasons.
I have made these cell lines competent as the control plasmid that comes with the topokit was successfully inserted, I know this as have grown these cells on chloramphenicol + amp plates, to select for rosetta/chaperone and my topo151 vector respectively.
The topo instructions say to use 5-10ng of plasmid, though that is with their BL21 cells that come with the kit. the protocol i used to make competent cells (CaCl2) may produce a different cell concentration and I was wondering if anyone knows if the concentration of plasmid is crucial? ie for some reason is it bad to add too much? I cant think why but I also cant think why no cells grew for these samples.
Thanks!
Sarah
May I ask, what is the transformation efficiency of your competent cells? How large is your plasmid?
Since your cells are chemical competent cells, more DNA is better. More DNA means more transformed colonies. What tends to be the problem of using too much DNA, is that one gets too many colonies and thus end up with a plate where it is nearly impossible to pick a single isolate colony.
Also does you enzyme have any know toxicity problems?
You mention codon usage... so just to clarify is the problem lack of expressed protein or lack of colonies carrying your expression plasmid.
perneseblue on Jul 21 2010, 08:57 PM said:
Since your cells are chemical competent cells, more DNA is better. More DNA means more transformed colonies. What tends to be the problem of using too much DNA, is that one gets too many colonies and thus end up with a plate where it is nearly impossible to pick a single isolate colony.
Also does you enzyme have any know toxicity problems?
You mention codon usage... so just to clarify is the problem lack of expressed protein or lack of colonies carrying your expression plasmid.
im unsure of the transformation efficiency, I made the cells competent myself...
I will try again with more plasmid. The enzyme has never been characterized before, im not even sure if its a functional enzyme.
I initally tried to clone the plasmid into standard BL-21 competent cells, but no protein was expressed. So i thought that might be a problem with codon usage as the enzyme appears to use many rare codons. So then I made some competenet rosetta (+codon) and chaperone (over expression of GroEL etc) cells. but having a problem getting my plasmid in here,
I will just add a lot of plasmid and hope for the best!
sfl40 on Jul 22 2010, 03:00 PM said:
perneseblue on Jul 21 2010, 08:57 PM said:
Since your cells are chemical competent cells, more DNA is better. More DNA means more transformed colonies. What tends to be the problem of using too much DNA, is that one gets too many colonies and thus end up with a plate where it is nearly impossible to pick a single isolate colony.
Also does you enzyme have any know toxicity problems?
You mention codon usage... so just to clarify is the problem lack of expressed protein or lack of colonies carrying your expression plasmid.
im unsure of the transformation efficiency, I made the cells competent myself...
I will try again with more plasmid. The enzyme has never been characterized before, im not even sure if its a functional enzyme.
I initally tried to clone the plasmid into standard BL-21 competent cells, but no protein was expressed. So i thought that might be a problem with codon usage as the enzyme appears to use many rare codons. So then I made some competenet rosetta (+codon) and chaperone (over expression of GroEL etc) cells. but having a problem getting my plasmid in here,
I will just add a lot of plasmid and hope for the best!
MIght it not be possible to conduct a test of the cells competency. Transform 10pg of plasmid which gives antibiotic resistance, and count how many colonies you get after plating a portion of the recovery culture. From there you should be able to work out how competent the cells were per ng DNA.
Hey,
what kind of chaperon cells you are using?? Where from you got those cells?
I am also struggling with protein expression.
I am planning to use chaperon and orgami cells.
Let me know if you have any idea//////
Thanks
macroman on Fri Jul 30 21:02:09 2010 said:
Hey,
what kind of chaperon cells you are using?? Where from you got those cells?
I am also struggling with protein expression.
I am planning to use chaperon and orgami cells.
Let me know if you have any idea//////
Thanks
sorry it has taken me so long to reply, i thought id wait to see if my plan worked- which it didnt
but i was using rosetta cells and some other cells that over-express chaperone proteins (I have asked my supervisor to get back to me on the name/company that makes these as I am not sure) to try and encourage expression of my protein but it didnt work!
Are you using the topo kit to induce expression from a lacz promoter? I have got the control system working, but not my protein.