I do not think the lack of colonies is due to your toxic gene it might be due a bad transformation since expression from pet15b requires T7 RNA polymerase which is not present in dh5 alpha (unless you have a very toxic gene then the readthrough transcription from the upstream bla promoter could cause toxicity and you will not get any colonies,if that the case you need to switch to a vector in which the promoter orientation is flipped I think that this got fixed in the newer generation of expression vectors or you will need to ligate a terminator upstream of the T7 promoter), also dh5 alpha is not a good strain for protein expression usually a B ecoli strain is used.

Your best option is to use BL21(DE3)pLysE which cotains a plasmid encoding T7 lysozyme that inhibits T7 RNA polymerase you can also use BL21(DE3)pLysS but the pLysS plasmid produces a lower level of T7 lysozyme thus less control.

Here is a link to the cells:

https://www.thermofisher.com/order/catalog/product/C656503

Best of luck!

Hanna

I do not think the lack of colonies is due to your toxic gene it might be due a bad transformation since expression from pet15b requires T7 RNA polymerase which is not present in dh5 alpha (unless you have a very toxic gene then the readthrough transcription from the upstream bla promoter could cause toxicity and you will not get any colonies,if that the case you need to switch to a vector in which the promoter orientation is flipped I think that this got fixed in the newer generation of expression vectors or you will need to ligate a terminator upstream of the T7 promoter), also dh5 alpha is not a good strain for protein expression usually a B ecoli strain is used.

Your best option is to use BL21(DE3)pLysE which cotains a plasmid encoding T7 lysozyme that inhibits T7 RNA polymerase you can also use BL21(DE3)pLysS but the pLysS plasmid produces a lower level of T7 lysozyme thus less control.

Here is a link to the cells:

https://www.thermofisher.com/order/catalog/product/C656503

Best of luck!

Hanna

Hi,

DH5alpha is cloning strain as Hanna mentioned you are not getting the colonies due to ligation Efficiency or your competence efficiency is very poor.

If you want to express toxic protein C41(DE3), C43(DE3), C41(DE)pLysS, and C43(DE3)pLysS is best strain to express toxic proteins.

Dear Sandeep

I’m agree with Hanna that you result cannot be due to the expression of your protein in DH5aplha because this cells do not express the T7 polymerase that it is necessary for protein expression with the t7 promoter.

Probably you need to optimize your cloning in some steps.

However, regarding your question about E. coli strains well repressed before induction and therefore more used with toxic gene you can try plyss strains or pLEMO strains. Im my experience sometimes you can solve the problem also using a glucose rich media with BL21(DE3) (e.g by supplementing LB with 1% of glucose or using a chemically defined media, or using the Enpresso B media (you can find information about it on my blog: PROTEOCOLL http://proteocool.blogspot.com/) but each protein as its own history.

For example, also if your protein is a toxin is also possible that its expression in E. coli will not result toxic for E.coli. Some years ago we successfully expressed the clostridium toxin B catalytic domain (tcdB-GT) in E.coli (https://www.ncbi.nlm.nih.gov/pubmed/23716610) with-out any problem during the cloning and expression probably because the toxin to be active need to incorporate Mn and UDP-glucose that was not present in the E.coli cytoplasm in enough amount and therefore it was expressed as apo- inactive form.

On the contrary we never succeeded in expression of the toxinA catalytic domain in E.coli, but also in this case, toxinA did not affect the E.coli transformation or growth rate but it was just expressed in a truncated form and only Brevibacillus was able to express it in the correct form https://www.ncbi.nlm.nih.gov/pubmed/22747490)

Good luck

Manuele

Is the toxin part of some prokaryotic toxin-antitoxin system?

@Peter Kysel, No, the toxin part is not prokaryotic toxin-antitoxin system.

I think this strain is good for your experiment "T7 Express lysY/Iq Competent E. coli (High Efficiency)" or "T7 Express lysY Competent E. coli (High Efficiency)"

https://www.neb.com/products/c3013-t7-express-lysyiq-competent-e-coli-high-efficiency#Product%20Information

https://www.neb.com/products/c3010-t7-express-lysy-competent-e-coli-high-efficiency#Product%20Information

I agree with Hanna and and Kannan, the DH5alfa is not your problem. If you have problems on next stage (protein expression) i recommend the C41 and C43 (DE3) strains, that are recommended to toxic and membrane proteins