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Toxic Plasmid?? Toxic Insert??


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#1 sandiablo

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Posted 29 March 2009 - 09:27 PM

I work with two family members of human glycine transporters - Glyt1b and Glyt2a. Both are cloned into the same vector - a derivative of pBSK- that has been modified to facilitate in vitro mRNA production (under the control of the T7 promoter) suitable for subsequent expression in oocytes...that is to say, this is NOT a bacterial expression system...

However, when growing these clones in DH5alpha cells, Glyt2a grows great - large bacterial pellets and corresponding high yields of plasmid DNA, whereas Glyt1b is total crap! Low growth and low yields. I suspected Glyt1b may have picked up a mutation in the F1 origin, so I cloned the insert back into the vector that had previously housed 2a with the same results - crap growth and yield. I typically recover only 4 ug of 1b from 10 mL of LBA culture (and before you blast my minis, I get 15-20 ug from 5 mL of 2a done side by side).

I have always heard rumors about DNA inserts that are toxic to host cells, but I have never encountered one - and certainly not one that would be toxic in an environment that is not actively promoting protein expression in the bacteria. Does anyone have any experience with anything like this? I've tried growing in TB and growing for 24 hours, with no increase in culture turbidity. Would it grow better in another strain? Different temperature? Different vector? Is T7 'leaky' in DH5alpha cells? Could one glycine transporter be more toxic than a closely related family member? HOW??? Is there a resource for this besides word of mouth and tall tales told over cocktails?

Captain, I've got to have more DNA!

#2 molgen

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Posted 30 March 2009 - 12:37 AM

I have always heard rumors about DNA inserts that are toxic to host cells, but I have never encountered one - and certainly not one that would be toxic in an environment that is not actively promoting protein expression in the bacteria. Does anyone have any experience with anything like this? I've tried growing in TB and growing for 24 hours, with no increase in culture turbidity. Would it grow better in another strain? Different temperature?

Changing the growth medium and/or the temp. shouldn't change the expression of the gene in the plasmid.

Different vector?

Can't hurt to try.

Is T7 'leaky' in DH5alpha cells?

T7 promoter comes from a bacteriophage and therefore is 'leaky'. It's a very strung bacterial promoter in all E.coli strains.

Could one glycine transporter be more toxic than a closely related family member? HOW??? Is there a resource for this besides word of mouth and tall tales told over cocktails?

If the insert is not in frame, the result can be toxic. Check that your inserts are in the same frame.

#3 phage434

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Posted 30 March 2009 - 04:31 AM

The T7 promoter is not "leaky" in a strain which does not contain the T7 RNA polymerase gene, usually carried on the (DE3) insertion. Common cloning strains do not contain this gene. Much more likely is that the problematic gene has a cryptic promoter or other toxic sequence. I would first try growing at a reduced temperature. You might also try chloramphenicol amplification, which would turn off protein production and enhance plasmid production.




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