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Problem with possible leaky expression in cloning

leaky expression top 10 cloning toxic protein

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4 replies to this topic

#1 Meg P. Anula

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Posted 29 December 2015 - 03:42 AM

Hi guys,

 

I am transforming a recombinant plasmid into top10 e.coli strain, to replicate more plasmid so I can use it in LAB bacteria.

 

And several observations makes me believe that there could be some leaky expression of the protein even in top 10 cell that causing some toxicity.

 

When I change the incubation temperature to 30 instead of 37, I manage to get positive colonies.

 

Now 

 

I am trying to transform another recombinant plasmid that express bigger protein, the temperature strategy alone doesn't solve the leaky expression problem anymore. I am wondering if I should grow it at even lower temperature. Or is there other tricks I may try to solve the toxicity issue? 

 

Thanks.



#2 labtastic

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Posted 30 December 2015 - 10:23 AM

Is it a lac- or arabinose-based expression plasmid, e.g. pET/pBAD? If so, you could try to adding in some glucose (~0.5%) to help repress expression.

 

Depending on what plasmid you are using, you might be able to clone your insert into a lower copy expression plasmid (like with a p15a or CDF origin) which might minimize basal expression.

 

Or is it possible you are using too much antibiotic for the plasmid you are growing? 

 

I doubt it would help but maybe you can try a different cloning strain of Ecoli, like DH5alpha.


Edited by labtastic, 30 December 2015 - 10:32 AM.


#3 Meg P. Anula

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Posted 31 December 2015 - 01:24 AM

Is it a lac- or arabinose-based expression plasmid, e.g. pET/pBAD? If so, you could try to adding in some glucose (~0.5%) to help repress expression.

 

Depending on what plasmid you are using, you might be able to clone your insert into a lower copy expression plasmid (like with a p15a or CDF origin) which might minimize basal expression.

 

Or is it possible you are using too much antibiotic for the plasmid you are growing? 

 

I doubt it would help but maybe you can try a different cloning strain of Ecoli, like DH5alpha.

 

I am using a pretty new plasmid, I don't think it is a lac or arabinose operon based plasmid. My project revolves around the study of this new plasmid which is from lactic acid bacteria, so I can't use other plasmid. But I'll give a try on the glucose tips.

 

I might use too much antibiotics tho. As there's often background after incubation for more than a day or two so I added more this time. I am using erythromycin which is only bacteriostatic. 

 

But is too much antibiotic a problem? As long as it contains the recombinant plasmid the bacteria should then be able to grow right?



#4 labtastic

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Posted 31 December 2015 - 05:40 AM

So is this a natural plasmid taken right from the lactic acid bacteria and put into Ecoli? If that is the case, do you know that it has an origin of replication that is compatible with Ecoli?

 

Indeed it is possible to use too much antibiotic. Seems like for erythromycin the working concentration should be around ~20ug/ml? If you use too much you will overwhelm the ability of the resistance protein to degrade the antibiotic before it binds and inhibits protein synthesis.



#5 Meg P. Anula

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Posted 02 January 2016 - 08:47 PM

So is this a natural plasmid taken right from the lactic acid bacteria and put into Ecoli? If that is the case, do you know that it has an origin of replication that is compatible with Ecoli?

 

Indeed it is possible to use too much antibiotic. Seems like for erythromycin the working concentration should be around ~20ug/ml? If you use too much you will overwhelm the ability of the resistance protein to degrade the antibiotic before it binds and inhibits protein synthesis.

 

Yes it has compatible ORI. I managed to clone one gene in top10 by incubating in 30c for 2 days. Now there's another gene which is bigger (and express bigger protein), I've tried few strategies but it just can't be transformed in. The ligation shouldn't be a problem. I suspect the bigger protein has bigger toxicity on the cell.







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