10 replies to this topic

[nescardoso]

Hey!

I'm trying to culture some BL21 (DE3) to express my protein...I transformed the bacteria, everything worked...I had many individual colonies. I picked one, inoculated in 5 ml LB medium supplemented with kanamycin (30µg/ml), they grow fine (I even made a glycerol stock), but when I inoculated 1L LB medium (as well containing kanamycin (30µg/ml)), the bacteria didn't grow!!! I repeated everything, and still no growth when I inoculate the big flask!

Anyone here can shed a light on this??? (because I thought of everything and I cannot find an explanation...)

Thanks in advance!

Inês

[prabhubct]

Did you tried to grow again in 5 ml LB O/N did it grow? and then inoculate fresh culture in 1 L LB.
I would have used both chloramphenicol and kanamycin initially in selection. BL21 is chloramphenicol resistant.

Edited by prabhubct, 06 September 2012 - 05:31 AM.

[nescardoso]

Thanks for your quick reply.

The preculture in 5ml I didn't grow O/N...only 4h, and then inoculated 1L LB and no grow again....

But I still don't understand how can they grow in 5ml of LB and not in 1L??? with the exact same amount of antibiotic...

[prabhubct]

colony you picked may be satellite colony.

[nescardoso]

I picked an individual, well-isolated colony, ....both times!!! I would understand this happening once (maybe that colony wasn't very healthy or was a satellite colony...), but twice?!...

[prabhubct]

Once your plate has satellite colony your chances of picking wrong increases. satellite colonies grow in more number than positives.

[Akdor]

To be sure that you are not picking a satellite you can double the amount of Kanamycin and keep the pre-culture O/N. I would go further only in the O/N pre-culture grows, otherwise... false positive and try luck again.

[ascacioc]

@prabhubct: BL21(DE3) is not chloramphenicol resistant!!!! BL21(DE3)pLysS is the chloramphenicol resistant one. or the CodonPlus. or BL21(DE3)Gold is Tet resistant. BL21(DE3) is empty of resistances. genotype: F^– ompT gal dcm lon hsdS_B(r_B^- m_B^-) λ(DE3 [lacI lacUV5-T7 gene 1 ind1 sam7 nin5]). See? No cm^r anywhere.

Anyhow. Coming back to the original: what is the volume of the flask that you are growing 1 L of culture in? (1L should be grown in 5 L flask: there is this rule that you don't have enough aeration unless you have just 1/5 or 1/6 of the total volume filled with media) what is the shaking speed (you need to shake 150 rpm, depending of the shaker to have enough aeration)? how much from preculture are you inoculating in the big flask? (~10 mL (I do 4 mL usually) from overnight preculture should be inoculated in 1 L) (and yes you need a preculture, you cannot inoculate 1 colony in 1 L because the lag phase will be infinity or several days)

Andreea

[nescardoso]

I'm doing a preculture in 5ml, and usually I just let it grow for 4h (not O/N) and then inoculate 2x 500 ml of LB medium in 2 flasks of 3L each...I'm shaking at 220rpm, 37C. I did this protocol to express other protein (same E.coli strain, but different vector) and everything went fine....

Maybe I need to do a preculture O/N and then inoculate 1L....I'll try that!


Thanks for the help!

Inês

Edited by nescardoso, 06 September 2012 - 11:57 PM.

[prabhubct]

@prabhubct: BL21(DE3) is not chloramphenicol resistant!!!! BL21(DE3)pLysS is the chloramphenicol resistant one. or the CodonPlus. or BL21(DE3)Gold is Tet resistant. BL21(DE3) is empty of resistances. genotype: F^– ompT gal dcm lon hsdS_B(r_B^- m_B^-) λ(DE3 [lacI lacUV5-T7 gene 1 ind1 sam7 nin5]). See? No cm^r anywhere.

my mistake.

ascacioc, on 06 September 2012 - 03:06 PM, said:

Anyhow. Coming back to the original: what is the volume of the flask that you are growing 1 L of culture in? (1L should be grown in 5 L flask: there is this rule that you don't have enough aeration unless you have just 1/5 or 1/6 of the total volume filled with media) what is the shaking speed (you need to shake 150 rpm, depending of the shaker to have enough aeration)? how much from preculture are you inoculating in the big flask? (~10 mL (I do 4 mL usually) from overnight preculture should be inoculated in 1 L) (and yes you need a preculture, you cannot inoculate 1 colony in 1 L because the lag phase will be infinity or several days)

Andreea

when I pick one colony and put in 100ml culture, If its positive it grows well. I supposed same should work for 1 liter culture too may be less growth but growth must be there. 10 times volume should not make much difference.

nescardoso, on 06 September 2012 - 11:56 PM, said:

I'm doing a preculture in 5ml, and usually I just let it grow for 4h (not O/N) and then inoculate 2x 500 ml of LB medium in 2 flasks of 3L each...I'm shaking at 220rpm, 37C. I did this protocol to express other protein (same E.coli strain, but different vector) and everything went fine....

Maybe I need to do a preculture O/N and then inoculate 1L....I'll try that!


Thanks for the help!

Inês

Yes you need to use overnight culture if you are using it for protein expression

[phage434]

I would double check that your 1 liter LB medium is what you think it is.  Use the same bottle of the same medium for picking colonies off your plate, so that there is no ambiguity about the composition and antibiotic status of your medium.  Use a large enough flask, as ascacioc suggests.