Do I have to add chloramphenicol to the LB ?? when using this particular expression cells ? I am going to express bacterial proteins , and i have inserted them individually into the vector pET-28a (+) GenScript .
BL21(DE3) pLysE Chemically Competent Cells ? the protocol from Sigma does not show up any sentence or words regarding this ?
https://www.sigmaaldrich.com/technical-documents/protocols/biology/bl21-chemically-competent-cells.html
if you want to use pLysE then yes you have to include CAM + selection antibiotic for your own plasmid. It does say in the manual just indirectly "plate transformation reaction directly onto LB agar plates containing the appropriate antibiotic".
Yes but nothing mentioned about cholrampheicol ? In the protocol from Sigma ? Thats why i am confused
The type of antibiotic resistance gene the vector pET-28a (+) has , i.e. kanamycin , ampicillin or chloramphenicol should be added both to the plate and LB. IPTG should also be added to the LB for protein induction. Isopropyl β-D-1-thiogalactopyranoside (IPTG) also known as lad-y) is a molecular biology reagent. This compound is a molecular mimic of allolactose, a lactose metabolite that triggers transcription of the lac operon, and it is therefore used to induce E.coli protein expression where the gene is under the control of the lac operator.
when they say appropriate antibiotic they mean CAM + selection antibiotic. Not the best way to phrase it I guess but you should add both.
Dear brother Irfan,
Yes the anti is kanamycin , all is ready for the experiment ,, but I am not sure do I have to add chloramphenicol or not ? as long as its chemical cells ..
Hello Mohammed Al-Nussairawi ,
Hanna Alalam has succinctly stated what you need to consider doing...
... BL21(DE3)pLysE, just like the similar E. coli strain BL21(DE3)pLysS, offers tight transcriptional control over basal leaky expression of the protein of interest (POI).
DE3 basically indicates that the strain expresses IPTG-inducible T7 RNA pol.
pLysE, similar to pLysS, is a plasmid that expresses low level of T7 lysozyme which is a natural inhibitor of T7 RNA pol and thus helps to reduce leaky expression by minimising basal T7 RNA pol activity in the absence of IPTG. (pLysE just has a better reduction of leakage compared to pLysS because of slightly higher level of T7 lysozyme expression, but the former would perhaps have a lower level of POI expression). These plasmids (pLysE and pLysS) also carry the chloramphenicol resistance gene.
...
So, you should add chloramphenicol into your media (in addition to kanamycin for the pET-28a) to maintain the pLysE plasmid. Without chloramphenicol, your POI would still be well expressed though, if not better, which is fine if you are not concerned about leaky expression, but the pLysE plasmid would be lost eventually and you would basically end up with the parent BL21(DE3) E. coli strain. However, control over leaky expression is actually the point of using the pLysE strain rather than the parent BL21(DE3) strain...
Dear Mohammed Al-Nussairawi
pLysE strain containing a compatible chloramphenicol-resistant plasmid that provides a small amount of T7 lysozyme, a natural inhibitor of T7 RNA polymerase.The lysozyme provided by pLysE can substantially increase the lag time and reduce the maximum level of expression of target genes upon induction of T7 RNA polymerase. This damping effect on expression is sufficient that cells containing a target gene whose product is relatively innocuous can continue to grow indefinitely in the presence of IPTG, a property that may be useful in some circumstances. However, the this promoter is relatively weak , and most transcription should be from aathe much stronger promoter used in PET28a. So alone kanmycin is enough whether you use chloramphenicol or not, it has no effect on your yield in this case
Many thanks for your answers ... I already did it and got nice colones with kanamycin only but not much only 4-5 but all of them contain the plasmids with the inserts ....however, the IPTG induction time was 10 hours with 0.9-1 OD ..but went well ..
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