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Problems with overnights

Overnights

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5 replies to this topic

#1 Smog187

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Posted 22 January 2013 - 06:22 AM

Hello,

I currently cloned in a 5kb gene into the PGEX vector (5kb), so now my current plasmid is 10kb. After cloning, I transformed the plasmid into stellar competent cells to create a DNA library. I then did a miniprep (and also confirmed by restriction digest that my insert was still there), then I transformed the plasmid into Rosetta Competent Cells (which are BL21 derivatives) and here is where my problems are starting.

So I've come to discover that it takes about 48 hours for my overnights to grow. This also happens when I finally do get a 5mL overnight to grow, then transfer that into a 50mL overnight. I'm worried that the bacteria that start growing around 48hours are growing because the antibiotics have degraded by this point (Ampicillin and Chloramphenical).

Does anyone have any experience with this? Is 48 hours to grow an overnight sound about right? Also why can an overnight of my plasmid in Stellar Competent cells grow "normally" in an overnight, but the plasmid in Rosetta Competent Cells takes forever to grow?

Thanks!

#2 HOYAJM

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Posted 22 January 2013 - 07:42 AM

You should worry that the antibiotics have degraded (they likely have). When it takes that long for your culture to grow, expression becomes very tricky. How will you grow your bacteria to a specific OD (unless you monitor the culture for 24 hours)? Anyways, try to see if you can retransform your plasmid into regular BL21 cells and repeat. If this fixes the problem, you just had bad rosetta cells. I have used both Rosetta pLysS cells and BL21 star DE3 cells for the same plasmid with nearly identical results for two proteins.

In short, something is not right since the stellar cells grow fine. I doubt the gene is toxic since your normal competent cells grow fine. Just make sure your transformation into the Rosetta cells worked (are you 100% sure your plasmid is in there?).

#3 Schevchen

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Posted 20 February 2013 - 04:41 AM

Ampicillin might be a problem, because it is not one of the most stable antibiotics. But chloramphenicol is (if you don't put it under UV light all the time). And I guys you grow them in an incubator. What you could also try is to lower the antibiotic concentrations and see if they grow better, you might have to optimise the growth with this particular plasmid.

#4 chavasuri

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Posted 25 March 2014 - 09:37 AM

Hello, 

I  cloned in a 1566bp gene into the PGEX-5x-3 vector (1.56 kb).  After cloning, I transformed the plasmid into Dh5 alpha competent cells  (and also confirmed by restriction digest that my insert was still there), then I transformed the plasmid into Rosetta Competent Cells (which are BL21 derivatives) and here is where my problems are starting. 

So I've come to discover that it takes about 48 hours for my overnights to grow. This also happens when I finally do get a 5mL overnight to grow, then transfer that into a 50mL overnight. I'm worried that the bacteria that start growing around 48hours are growing because the antibiotics have degraded by this point (Ampicillin and Chloramphenical). 

Does anyone have any experience with this? Is 48 hours to grow an overnight sound about right? Also why can an overnight of my plasmid in Stellar Competent cells grow "normally" in an overnight, but the plasmid in Rosetta Competent Cells takes forever to grow? 

 

and also i tried with plyss and BL21-DE3 cells in that case cell death is observed after induction with IPTG(0.1, 0.5, 1.0), i repeated with different company IPTG and with positive controls (growing well after induction also). 

​only in presence of my gene of interest  cells are going to death phase or poor growth.  

 

can any one please address this problem....i appreciate your great help.  thanking you 



#5 Missle

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Posted 26 March 2014 - 04:03 AM

It sounds as if your Rosetta cells are only growing well after the antibiotic has been consumed and they have kicked the plasmid.  48 hrs is way too long.  You could check this by plating the cells on plates with and without the appropriate antibiotics and comparing the amount of colonies.

It also sounds as if your gene of interest is toxic to the cells.  When using the BL21 cells, did you get good overnight growth?  How about with the pLysS?

 

My advice would be to switch from ampicillin to carbenicillin (more stable) and - although it is logistically a pain - perform your expression from a fresh transformation.  You could try a lacIq mutant like Tuner and don't express for very long.

 

Good luck!



#6 labtastic

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Posted 28 March 2014 - 12:33 PM

So do your cloning cells grow just fine with the plasmid, and it is only when you move it into Rosetta's that you have growth issues? 

 

If that is the case, then my bet is that your expression product is toxic to the cells. In this case, you should try to find ways to minimize leaky expression why the cells are growing prior to induction. BL21ai cells were designed for this purpose by minimizing leaky expression by keeping the T7 expression system under the control of two sugars (lactose/IPTG plus arabinose) as opposed to traditional BL21 strains which require only lactose/IPTG. You could also try adding in a small amount of glucose to repress expression (<0.05%) while the cells grow. Or try autoinduction media, which has glucose in it for intial growth but when it gets depleted the cells take in the lactose, which induces expression.

 

Or just try a different vector altogether.






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