15 replies to this topic

[chn09]

Hello everyone,
Im trying to express a his tagged protein in E.coli BL21 DE3. I prepare the starter culture by inoculation of a single colony of the clone im 50 ml LB . I leave it overnight, next morning i use it to inoculate another set of LB media. Does any one know how long the starter culture should be grown?. i ve seen literature that recommends growth of starter cultures upto 16 hrs. Pls help

[KAUSHIK THAKKAR]

chn09, on Sep 16 2009, 12:30 PM, said:

Hello everyone,
Im trying to express a his tagged protein in E.coli BL21 DE3. I prepare the starter culture by inoculation of a single colony of the clone im 50 ml LB . I leave it overnight, next morning i use it to inoculate another set of LB media. Does any one know how long the starter culture should be grown?. i ve seen literature that recommends growth of starter cultures upto 16 hrs. Pls help

Hi,
Typically you can grow the starter culture for about 16-20 hrs.
And may be inoculate about 1/10th volume of the medium, next day...!!

[Wolfgang]

chn09, on Sep 16 2009, 02:00 PM, said:

Hello everyone,
Im trying to express a his tagged protein in E.coli BL21 DE3. I prepare the starter culture by inoculation of a single colony of the clone im 50 ml LB . I leave it overnight, next morning i use it to inoculate another set of LB media. Does any one know how long the starter culture should be grown?. i ve seen literature that recommends growth of starter cultures upto 16 hrs. Pls help

I usually inoculate 10ml culture in a 50ml tube overnight(approxiamately 18 hours), next day transfer 5ml into 100ml prewarmed media.

[ram]

KAUSHIK THAKKAR, on Sep 16 2009, 12:55 AM, said:

chn09, on Sep 16 2009, 12:30 PM, said:

Hello everyone,
Im trying to express a his tagged protein in E.coli BL21 DE3. I prepare the starter culture by inoculation of a single colony of the clone im 50 ml LB . I leave it overnight, next morning i use it to inoculate another set of LB media. Does any one know how long the starter culture should be grown?. i ve seen literature that recommends growth of starter cultures upto 16 hrs. Pls help

Hi,
Typically you can grow the starter culture for about 16-20 hrs.
And may be inoculate about 1/10th volume of the medium, next day...!!

This is related to my another post...why to use only 1/10th volume of starter culture? Instead can't it be grown for less time and the whole starter culture used? If the starter culture is grown for 16-20 hrs, wont it cross the log phase (OD 0.6)?

[Vini]

ram, on Nov 4 2009, 10:55 AM, said:

KAUSHIK THAKKAR, on Sep 16 2009, 12:55 AM, said:

chn09, on Sep 16 2009, 12:30 PM, said:

Hello everyone,
Im trying to express a his tagged protein in E.coli BL21 DE3. I prepare the starter culture by inoculation of a single colony of the clone im 50 ml LB . I leave it overnight, next morning i use it to inoculate another set of LB media. Does any one know how long the starter culture should be grown?. i ve seen literature that recommends growth of starter cultures upto 16 hrs. Pls help

Hi,
Typically you can grow the starter culture for about 16-20 hrs.
And may be inoculate about 1/10th volume of the medium, next day...!!

This is related to my another post...why to use only 1/10th volume of starter culture? Instead can't it be grown for less time and the whole starter culture used? If the starter culture is grown for 16-20 hrs, wont it cross the log phase (OD 0.6)?

I grow my starter culture of 50 mL for about 12 hrs. and then use the whole for incoulating a 1L medium....

[ram]

DRN, on Nov 3 2009, 11:41 PM, said:

ram, on Nov 4 2009, 10:55 AM, said:

KAUSHIK THAKKAR, on Sep 16 2009, 12:55 AM, said:

chn09, on Sep 16 2009, 12:30 PM, said:

Hello everyone,
Im trying to express a his tagged protein in E.coli BL21 DE3. I prepare the starter culture by inoculation of a single colony of the clone im 50 ml LB . I leave it overnight, next morning i use it to inoculate another set of LB media. Does any one know how long the starter culture should be grown?. i ve seen literature that recommends growth of starter cultures upto 16 hrs. Pls help

Hi,
Typically you can grow the starter culture for about 16-20 hrs.
And may be inoculate about 1/10th volume of the medium, next day...!!

This is related to my another post...why to use only 1/10th volume of starter culture? Instead can't it be grown for less time and the whole starter culture used? If the starter culture is grown for 16-20 hrs, wont it cross the log phase (OD 0.6)?

I grow my starter culture of 50 mL for about 12 hrs. and then use the whole for incoulating a 1L medium....

Hi DRN
Did u anytime take OD of your 12 hrs grown starter culture...Did u get proper expression after doing so? If yes, can u just post a snapshot of SDS-PAGE having your sample and negative control loaded on the same gel?

Edited by ram, 04 November 2009 - 03:45 AM.

[Vini]

ram, on Nov 4 2009, 03:06 PM, said:

DRN, on Nov 3 2009, 11:41 PM, said:

ram, on Nov 4 2009, 10:55 AM, said:

KAUSHIK THAKKAR, on Sep 16 2009, 12:55 AM, said:

chn09, on Sep 16 2009, 12:30 PM, said:

Hello everyone,
Im trying to express a his tagged protein in E.coli BL21 DE3. I prepare the starter culture by inoculation of a single colony of the clone im 50 ml LB . I leave it overnight, next morning i use it to inoculate another set of LB media. Does any one know how long the starter culture should be grown?. i ve seen literature that recommends growth of starter cultures upto 16 hrs. Pls help

Hi,
Typically you can grow the starter culture for about 16-20 hrs.
And may be inoculate about 1/10th volume of the medium, next day...!!

This is related to my another post...why to use only 1/10th volume of starter culture? Instead can't it be grown for less time and the whole starter culture used? If the starter culture is grown for 16-20 hrs, wont it cross the log phase (OD 0.6)?

I grow my starter culture of 50 mL for about 12 hrs. and then use the whole for incoulating a 1L medium....

Hi DRN
Did u anytime take OD of your 12 hrs grown starter culture...Did u get proper expression after doing so? If yes, can u just post a snapshot of SDS-PAGE having your sample and negative control loaded on the same gel?

Hi Ram
I never take the od... .......12 hrs. is an approximate time, sometimes i keep it for more time (too lazy 2 come early in the morning).........i mean its kind of routine stuff, so i don't bother with taking the od. And yes, no matter, whether i keep for 12 hrs. or 14 hrs. (in fact, i hv also kept the culture just for 7 hrs.), I do get protein expression....As for the gel pic, i don't hv any pics with the negative control . Simply because, i would hv already checked the expression on mini-scale with the negative control. Hope this helps....

[ram]

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!

Edited by ram, 04 November 2009 - 07:25 AM.

[Vini]

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)????

[ram]

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?

[Vini]

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  

[ram]

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.invitro...OPOBrochure.pdf. So in conclusion, there is no separate vector available.

Edited by ram, 05 November 2009 - 03:27 AM.

[Vini]

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.invitro...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!!!    

[ram]

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?

Edited by ram, 05 November 2009 - 03:50 AM.

[Vini]

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.........