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protein expression - Preparation of starter cultures (Sep/15/2009 )

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ram on Nov 5 2009, 02:16 PM said:

DRN on Nov 4 2009, 10:30 PM said:

ram on Nov 4 2009, 09:51 PM said:

Thanks for the info DRN!
nice to know that u get nice expression at such variable time of expression :) which vector r u using by the way?
Right now I am using Invitrogen pCR T7 TOPO TA Expression Kit. And I am not able to see any difference between SDS-PAGE patterns of induced and uninduced sample! :) :(



Hi Ram
I use pET vectors , mostly pET 21 ....I haven't used the TOPO TA expression kit, though.....If you are not able to see the difference, it could also be because of leaky expression....is there any difference b/w ur empty vector (induced) and clone (induced)????


My negative control was the uninduced sample aliquoted just before induction at 0.6 OD. Would there be leaky expression at this point also?
I had thought of doing control expression with an empty vector ...but as u know its a costly TOPO kit n i have the smallest one with only 10 reaction...so I cannot give away 1 only for empty expression...is there any other way?





yeah, thats right....that is what I mean by leaky expression. With some of our proteins also, we have had such case....of course, i compare with empty vector also to be sure of this. is it not possible for u to take 1 ul of the vector and make more copies of it?????..........i dunno, if i m asking something stupid :o

-DRN-

Ok! I had thought that leaky expression occurs only in the uninduced culture grown for about 5 hours after getting the OD. (Sample parallel to the induced culture at the time of harvest).
In TOPO expression system, vectors and ligase (replaced by Topoisomerase) are not provided separately. In fact, topoisomerase is covalently bound to the vector. You have to just add your insert to the "vector", place at RT for 5 min and transform! Thats the beauty of TOPO ligation kits (For details: http://tools.invitrogen.com/content/sfs/br...OPOBrochure.pdf. So in conclusion, there is no separate vector available.

-ram-

ram on Nov 5 2009, 05:54 PM said:

Ok! I had thought that leaky expression occurs only in the uninduced culture grown for about 5 hours after getting the OD. (Sample parallel to the induced culture at the time of harvest).
In TOPO expression system, vectors and ligase (replaced by Topoisomerase) are not provided separately. In fact, topoisomerase is covalently bound to the vector. You have to just add your insert to the "vector", place at RT for 5 min and transform! Thats the beauty of TOPO ligation kits (For details: http://tools.invitrogen.com/content/sfs/br...OPOBrochure.pdf. So in conclusion, there is no separate vector available.



Yeah, u r also right Ram....even that is leaky expression. but in ur present case also, there is a chance that same thing is happening....And, yes, I understand ur problem about vector now. Thanks for providing that info :( ........Hey, but "beauty" comes at a cost!!! :o

-DRN-

I forgot to write and observed it for the 3rd time that although there in no difference in protein profiles, the growth (as reflected in the turbidity and size of the pellet after harvesting) in induced culture is always less then that in uninduced . Why is it so? Does IPTG retard growth rate?

-ram-

ram on Nov 5 2009, 06:16 PM said:

I forgot to write and observed it for the 3rd time that although there in no difference in protein profiles, the growth (as reflected in the turbidity and size of the pellet after harvesting) in induced culture is always less then that in uninduced . Why is it so? Does IPTG retard growth rate?



I haven't observed so in my case....but if it is so, it could be because, the entire biosynthetic machinery in the bacteria will be switched off (or in a low mode) to divert resources for expressing the foreign gene in excess.....this might affect the growth rate.........

-DRN-

DRN on Nov 5 2009, 03:56 AM said:

ram on Nov 5 2009, 06:16 PM said:

I forgot to write and observed it for the 3rd time that although there in no difference in protein profiles, the growth (as reflected in the turbidity and size of the pellet after harvesting) in induced culture is always less then that in uninduced . Why is it so? Does IPTG retard growth rate?



I haven't observed so in my case....but if it is so, it could be because, the entire biosynthetic machinery in the bacteria will be switched off (or in a low mode) to divert resources for expressing the foreign gene in excess.....this might affect the growth rate.........


Thats what I also had thought of initially, but if would have been the case, there should have been the clear difference in the protein profiles which is not the case! I really wonder :D

-ram-
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