In a fast growth phase the E.coli genomic DNA is produced at a rapid rate and it takes time for all of it to be methylated and protected. New DNA will be digested by your overexpressed restriction enzyme and you will not get enough growth of the E.coli to get colonies.  This does not explain why the DH5alpha strain is not working. Is there any chance that there are other promoters e.g the antibiotic resistance gene that could carry over transcription into your gene?

I think your best bet is to use an arabinose inducible promoter to express the restriction enzyme and to induce expression when your cells are grown to late exponential phase or early stationary phase.  If possible clone the gene such that its promoter is the only one firing in that direction - ensure all other promoters on the plasmid are firing in the opposite direction.

Thanks for the answer. I will take into account the arabinose promoter, that´s for sure; however my main problem is that even with induction (because I am not adding IPTG to my culture or to my plates) I am not getting colonies. I am wondering if the restricction enzime is really toxic to my cells because maybe the basal expression of the T7 is already too much for the cell. 

I check already and there is not other promoters in the same direction , so expression should be only regulated by the T7.

Thanks a lot for the advices!

Probably you already did this, but if it is really a time problem: 

Are you using high copy number plasmids for the methylase? Did you increase the selection pressure for those clones? And are you using low copy numbers for the restrictase?

I'm also wondering why the DH5 doesn't work- you sure your ligation works fine?

The last possibility is to push the expression towards inclusion bodies, knowing that you will lose your easy purification approach, but sometimes its the only possible way 

Yes, my methylase is high copy number and my restrictase plasmid low copy. Ligtion is working fine for the rest of my clones, it is this one the one giving me problems! Other people from the lab have alredy tried to get this clone and they were never succesfull, so we should have toxic problems around!

As u sais, maybe inclusion bodies is the best solution!

Thanks again!

 I guess that Dh5 doesn´t work also because the basal expression of the T7 promoter (even without IPTG induction) is producing enought restrictase to kill the cells and this why I am not getting colonies after plating my transformant bacteria.

I will try to add Glucose to the media to avoid this leakage expression and to growth the bacteria at lower temperature for more days. We will see if I am lucky!

Hi, include glucose (2% v/v) in your agar plates and in liquid medium for repressing until you induce expression. You may also use BL21(DE3)plysS for additional repression by means of the inhibition of T7 RNA pol by lysozyme. For expression you can lower the temperature to 30-16oC and/or perform an overnight induction with no agitation. Good luck!

Thanks a lot for the answer. I am trying already adding glucose to the media in Lb and minimal media, to see what happen! Hopefully I will be lucky with BL21(de3) and I dont need to use the pLyss strain since the plasmid that I use to express the methylase is Cm resistance also!

Will keep u update, thanks a lot!

Hi Esther,

I am a bit confused-you are using BL21 for cloning transformations and miniprepping or only for expression? These cells are for expression only as they are not recA/endA and your plasmid may not be stable. If your methylase is in a T7- based plasmid then you need to change it for another promoter to use in DH5alpha as you need T7 polymerase (from DE3 strains) for transcription from the plasmid.

Gustavo's suggestion of glucose and/or Lys strains are excellent as I am pretty sure that a restriction enzyme will cause problems for your cells and needs to be blocked as well as possible before induction-the absence of glucose can make transformations impossible if you have leaky/toxic expression.

Do you know how common the restriction site is in E.coli genome and if it is present in your expression plasmids?

Auto-induction may also work as the cells are approaching saturating densities before induction so you will have more biomass with (hopefully intact expression plasmid) before the restriction enzyme is expressed.

Thanks a lot Nick.

I know BL21 is not a strain for cloning. I know that it should be using for protein expression. My main problem is that I have the restriction enzime clonned already in a plasmid with a pLac promoter. The problem comes because to purified the protein the person that is gonna do it want it with a His tag and a T7 promoter (in order to get higher expression), so I need to change the vector.

With this new vector I tried to clone in E.coli Dh5alfa but I never got colonies. I know that the T7 is not naturally expressed in Dh5alfa but I start to think that maybe the leack expression is just killing my cells, been the main reason for the absent of colonies. I am doing at the same time other clonning in the lab and the ligase is working perfectly. Moreover, when I am getting colonies in my clonning they are always possitivies because the vector has recicularized but without the restriction enzime inserted. So I guess that my gen is toxic and this is why I need to control a lot the expression under a good promoter.

Since I can not hange the promoter (T7) because it is the one that they require me for protein purification latter I thought that maybe although BL21(DE3) is not the best chooise for clonning can help me with the expression control. This is the only reason for choosing this strain as a clonning strain. Maybe it doesnt work neither but... I guess I should try!

As people mentioned me, I would add glucose to the media so like this maybe I can control better the T7. Will see if it is works, and of course try the pLySS strain that i read is the best one to control the leackage expression.

Thanks again for all ur advices. And If u think that they is a better strategy just to afford the problem, let me know please!

Esther

Dear Esther,

We are cloning and expressing one restriction enzyme right now, facing the exact problem like you did four years ago. May I know how did you overcome the problem?

Best regards!

Lumeng