Why do we generally prefer cells like DH5alpha or top10 for increasing plasmid amount and isolation? Why are we using BL21 for protein expression? Why not DH5alpha?
Hallo Sujeesh!
DH5 alpha has a recA mutation, so it does no heterologous recombination which ensures a higher insert stability . Additionally, it lacks some endonucleases which might digest the plasmids during the isoation procedure. DH5 alpha is additionally competent for blue-white screening.
BL21 on opposite is engineered for protein expression purposes. It is deficient in some proteases, so it will not digest recombinant proteins - or more precisely, it will do less. The often employed T7 RNA polymerase is expressed in the BL21 (DE3) strain from a genomic copy (introduced via a DE3 lysogen), upon induction by IPTG.
You will find more information on the genetic background e.g. on the homepage of New England Biolabs:
https://www.neb.com/products/c2527-bl21de3-competent-e-coli
https://www.neb.com/products/c2988-neb-5-alpha-competent-e-coli-subcloning-efficiency
or life technologies:
http://www.lifetechnologies.com/de/de/home/life-science/cloning/competent-cells-for-transformation/competent-cells-strains/dh5a-competent-cells.html?s_kwcid=AL!3652!3!35873371030!e!!g!!dh5%20alpha&ef_id=VHdbWgAABXIssxe-:20141127171154:s
https://www.lifetechnologies.com/order/catalog/product/C601003
Best regards,
Christian
Hallo Sujeesh!
DH5 alpha has a recA mutation, so it does no heterologous recombination which ensures a higher insert stability . Additionally, it lacks some endonucleases which might digest the plasmids during the isoation procedure. DH5 alpha is additionally competent for blue-white screening.
BL21 on opposite is engineered for protein expression purposes. It is deficient in some proteases, so it will not digest recombinant proteins - or more precisely, it will do less. The often employed T7 RNA polymerase is expressed in the BL21 (DE3) strain from a genomic copy (introduced via a DE3 lysogen), upon induction by IPTG.
You will find more information on the genetic background e.g. on the homepage of New England Biolabs:
https://www.neb.com/products/c2527-bl21de3-competent-e-coli
https://www.neb.com/products/c2988-neb-5-alpha-competent-e-coli-subcloning-efficiency
or life technologies:
http://www.lifetechnologies.com/de/de/home/life-science/cloning/competent-cells-for-transformation/competent-cells-strains/dh5a-competent-cells.html?s_kwcid=AL!3652!3!35873371030!e!!g!!dh5%20alpha&ef_id=VHdbWgAABXIssxe-:20141127171154:s
https://www.lifetechnologies.com/order/catalog/product/C601003
Best regards,
Christian
In very simple way DH5 alpha was designed for higher stability and transformation by genetic manipulations (mutations) while BL 21 for higher level of protein expression. The mutations that the DH5-Alpha strain has are: dlacZ Delta M15 Delta(lacZYA-argF) U169 recA1 endA1 hsdR17(rK-mK+) supE44 thi-1 gyrA96 relA1 (2). These mutations correspond to the distinct characteristics that make the DH5-Alpha strain excel in laboratory cloning procedures.
To my opinion, you can use BL21 for plasmid storage (it always worked in my case), but DH5 alpha are usually preferred for DNA transformation because they allow for blue/white screening. On the other hand, BL21 are used for protein expression because they lack many proteases which would cleave overexpressed proteins.
Christian Zerfass gave an outstanding answer to your question. Others reinforced the fact that BL21 cells are more prone to loss of plasmid, which will lower protein expression but can also cause other problems that DH5a are designed to avoid. I do not recommend storing your transformed BL21 cells in the freezer for later protein expression, but rather find that fresh transformants yield the best and most consistent protein expression. With one exception. An often overlooked source of a lot of information can be found in the paper from Bill Studier, who originally developed the BL21 and pET system and should be much more celebrated for these achievements in my opinion, in Protein Expression and Purification (2005) 41: 207–234.
Why can't you use DH5alpha for protein expression after the ccdB gene has be recombined with your protein of interest?
BL21 strain is generally used for protein over-expression purposes because it has more efficient transcriptional machinaries to give more protein.
@Pearl Choi, I believe ccdb gene is a toxin so it will kill the plasmid and prevent expression of your protein of interest.
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