Generally when we have to store E coli recombinant clone to prepare cell bank, we take in mind that frozen bacterial cells at -80°c could destroy cell membranes after a rapid thawing , thus it could breaks the cell membranes

Generally we have to frozen bacterial cell in 50% glycerol to ovoid both bacterial cell lysis and may be protein lysis and denaturation.

In fact your methods could destroy cells and therefore the induction of recombinant protein is blocked because you use a cell lysate and none intact bacteria so you haven’t promoter induction in that lysate.

However, bacterial clone are frozen at -80°c and rapidly thawed to succeed in colony PCR without preparing DNA plasmid by mini-prep. The DNA even in that lysate could be amplified.

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Thanks Mohamed Nejib Marzouki.

I stored cells in the Glycerol 50% (400ul) and LB Broth(600ul) mixture.

As per your first question is concerned i would like to ask what bacterial expression vector are you using for the expression because their are various bacterial vectors are there which are known of its leaky nature (one of which i am currently dealing with) mostly T7 promoter systems.

In addition, whenever you planning for an expression experiment you must get sequenced your recombinant plasmid and recheck for the insert and its frame by doing BLAST analysis.

Thanks Priyanka Namdev.

My vector is pET32a(+).

Your basal expression is probably protein from your bacteria that migrates as your target protein, as you see this band in the expression of digested plasmid. I always transorm the expression host with an empty vector/plasmid as a negative control and to see host proteins.

Thank you Anders O Magnusson.

Still Waiting for suggestions.

Hi Nii,

You didn't say what strain of E.coli you were using-if it's not recombinant deficient then you should not make glycerols in them as they can modify your plasmid over time depending on the plasmid and strain. This might give you protein the first time and either nothing or truncated proteins after that.

Store your DNA in a recombinant deficient strain and miniprep from those. Then for expression I prefer to do a fresh transformation into 'B' strains every time and use colonies from the plates within a week.

If you are using a lac promoter or the T7 system you can also block basal expression by the addition 1% glucose to your plates and media (unless you use AI media as Lisandra suggests-then add glucose only in your 'starter' culture and omit it when diluting your cells into the AI media). In the T7 system the LysS strains (again as Lisandra suggests) will also inhibit 'leaky' expression by inactivating the T7 polymerase.

Good luck

Thank you Lisandra Aguilar-Bultet and Nick S Berrow for your suggestions. i have used E. coli Rosetta-gami with pET32a(+) plasmid.

Still waiting for more suggestions.

Rosetta-gami should not be used to make glycerol stocks as they are unlikely to be stable. They are based on the B strain BL21.

Are your Rosetta-gami (DE3)? This is essential for pET vectors to work as this expresses the T7 polymerase.

My Strain is just Rosetta-gami. there is nothing after the name like DE3.

You cannot express from pET plasmids in those cells. The DE3 lysogen expresses the T7 polymerase after induction and this is necessary for transcription from the T7 promoter on all pET plasmids.

I hope it is just a case of bad labelling of your cells, if not you have to get the right E.coli strains for the T7 system.

Good luck

Thank you Nick S Berrow for your suggestion. do you have any publication about what expression system should be used or that introduce the host cells and their differences?

Thank you Lisandra Aguilar-Bultet.