We generally use. E.coli BL 21 for protein expression and E.coli DH 5a for cloning (amplification). But why can't we use BL 21 for both cloning and expression both? That would save a whole lot of time.
Dear Shubham
Generally BL21 are used for expression but not for cloning for several reason:
1) We prefer to use for cloning E.coli strain that are recA delete and therefore are less prone to perform DNA recombinantion and you have less risk of errors in the sequence of your final plasmid.
2) BL21generally result in less competence level respect other strains. For example using chemical competent protocoll (as Mgcl2/CaCl2) generally we obtain BL21 maximun at 10^6 cfu/ug while the DH5alpha, HK100 or other strains we can obtain with the same protocoll also 10^7cfu/ug easly and therefore you can have some troubles when performing difficult ligases (e.g insertion of long dna sequences)
3)As PAul-Antoinee suggest, if you are using BL21(DE3) with Plasmids as PET based on t7promoter you can have some risk of basal expression (protein expression also with out add IPTG) and in case you recombinant protein is toxic for the E.coli can make difficult the bacterial growth in the agar plates and than reduce drammatically the efficiency of your ligases.
Therefore you can certanilly try to use BL21 also fut you need yo consider the possible drawback of this approach.
In my experience to save time is more important choose a simple and modern cloning approaches )e.g PIPE cloning or LIC or Gateway) that allow you to avoid some steps (e.g pcr purification) respect the standard cloning with restrinction enzine/ligase than avoid this step that is quite easy and delay your work of one, maximun 2 days.
From NEB website:
The common expression strain BL21(DE3) is a poor choice for direct cloning, because its Endonuclease I activity may degrade plasmids after isolation, and its high basal T7 expression level may result in clone instability and/ or intolerance of toxic proteins
We have seen this also in our experiments in which plasmid was lost. There are plenty of other "good strains" out there to use to avoid unnecessary risks.
Ian R Wilkinson Hi, I met some problems in recent days that my plasmids were "lost"during plasmid miniprep using a column kit (Omega), and I know BL21(DE3) is not the suitable strain for plasmid isolation but in my experiment, I need to use this strain. May I ask you what plamids were lost in your experiment? Only the plasmid backbone will do. Thank you so much~
Hi Jiani Xing, as I recall the instability (or loss of expression) was found using pET series of plasmids with BL21 when stored as glycerol stocks... it was a long time ago!!
bw
Ian
Ian R Wilkinson Thanks for your reply. I used high copy number plasmid but got low plasmid content but I don't know where the mistakes were.
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