Hi Andy, just to follow up on Christian suggestion, which is exactly the way to go.
In case you are using an already pET based vector, simply try BL21-AI (https://www.thermofisher.com/order/catalog/product/C607003), which has the T7 RNA polymerase under arabinose promoter should do the trick. Also, 2% Glucose is a must in this kind of situation. I have been dealing with several toxic proteins (from the family of restriction enzymes :) ) and BL21-AI was a way to go. Please also have a look if your pET backbone has the extra copy of the lacI, which makes a difference in leakage expression. As a sum up: 1) try BL21-AI (for the induction of the target protein you will need both Arabinose (T7 RNA pol induction) and IPTG for the T7 promoter) 2) or BL21 with pLysS or pLysE both simple keep 2 % Glucose and also directly from trafo goto liquid culture in parallel of the plating approach. Let me know if you need any further tips. Nikolay Dobrev Postdoctoral Fellow @ Wilmanns group EMBL Hamburg, c/o DESY, Building 25A, Notkestraße 85, 22607 Hamburg, Germany T +49 40 89902 165 | M +49 173 684 0532 twitter.com/emblevents https://twitter.com/emblevents |http://facebook.com/embl.org | http://youtube.com/user/emblmedia Visit http://www.embl.org/events for a complete list of all EMBL events. > On 04/04/2022 10:32 AM Christian Roth <christianroth...@gmail.com> wrote: > > > Hi Andy, > have you tried another promotor? Arabinose is much tighter, just to be > sure that it is really not leaking. > > Cheers > Christian > > > On Mon, Apr 4, 2022 at 10:20 AM Andrew Lovering <a.lover...@bham.ac.uk > mailto:a.lover...@bham.ac.uk > wrote: > > > > > > Dear Board, > > > > > > > > Perhaps off-topic, but in the wider scope it’s relevant to many on > > here. > > > > > > > > We have a gene that we are able to clone, and propagate in DH5a etc > > non-expression cells (hence nucleotide sequence is non-toxic) > > > > > > > > But, when we attempt to transfer to an expression strain we get no > > colonies > > > > > > > > We have tried pLemo, Glucose addition, 30 degrees, C41/C43 and > > still no joy > > > > > > > > We’d welcome any suggestions here – it’s a fun protein > > > > > > > > Thanks > > > > Andy > > > > > > > > --------------------------------------------- > > > > To unsubscribe from the CCP4BB list, click the following link: > > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 > > > > > > > --------------------------------------------- > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 > ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
