I have my gene of interest in the plasmid pQE30. My gene of interest is AlaRS. I want to express the protein AlaRS . I obtained this plasmid from Addgene which says the plasmid grows in E. coli JM109. it was provided in a bacterial stab culture. I assume then it was provided in Jm109. I tried to isolate the plasmid and transform into BL21 cells but failed thrice. There were no colonies on transformed plate. This particular plasmid contains T5 promoter. Can you provide an explanation why I was unable to transform and how can I express the protein? Although i tried to express this protein with IPTG induction in the provided strain only but unable to identify whether my protein was expressed or not. Please take a look at the sds page gel image. I am supposed to get the protein at size- 98 kDa.
After you did your mini prep from the provided strain (JM109 presumably), did you look on a gel to be sure you actually have plasmid DNA? If you aren't getting colonies upon transformation then it is one of two things, either no plasmid or your competent cells are not good. You can do controls to check either of those out (use different competent cells and use a control plasmid for your BL21).
But note that expression levels from pQE30 are not going to be any better in BL21 than in JM109 except possibly that BL21 has fewer proteases.
Yes I did check them on gel and got positive results at expected size.
Then try the transformation again and also do it with some control plasmid just to confirm if your competent cells are good. If neither work, then you need to make new competent cells.
Are you using BL21 or BL21 (DE3)? Because BL21 alone does not express much lacI so your promoter will essentially be expressed constitutively and not inducible by IPTG. Even BL21 (DE3) is limiting in that respect but better.
So if your protein is at all toxic then you may not be getting transformants due to lethality. Many of the pET vectors carry the lacI gene on the plasmid so that there is plenty around, but pQE30 does not.
Dear Satarupa Deb Sinha
Are you sure that the problem is not related to the toxicity of the AlaRS protein for the cells. Since sometimes toxic protein, in presence of basal expression (expression before induction) may not allow the growth of the BL21 trasfromed cells in the agar plates.
A possible solution when you are using the T7 promoter is use the plyss strain or add glucose on the LB, since glucose is able to repress a little the T7 promoter.
i'm not an expert of T5 but it seems that glucose may do the same also in this promoter
https://www.avidity.com/showpdf.asp?N=028C14D4-8C05-4625-AF66-5782BDB5B054
so i suggest to you to add 1% of glucose in the LB-agar plates and see if it improve your trasfromation efficiency.
if yes, you can try to perform the culture by adding glucose in your pre.culture or otherwise using the enpressoB media, instead LB, which is based on glucose feed batch and therefore show very good repression before the IPTG induction
in the follwing link you can find some more infromation about if;
https://www.blogger.com/video.g?token=AD6v5dwU8Sl-43BfVTSeIRFBcQiWXAeg80Ud5UCxiXqUj4AFUO7p1BOHHmV4NiV0MY32A41mTgUH2-nyouDVBhcVFOuGc1lJ5_1WgP3RrsvakB6iJHCW_JOnrD8kIeiUSimWYahOs-OP
good luck
Manuele
Hi Satarupa, what protocol are you using for transformation, I recently did the transformation of BL-21 with a plasmid (kanamycin) and used the concentration around 13ng/10 microlitre of bacteria and it worked. And also you the volume of your plasmid should be between 1to5 microlitre.
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