Re: [ccp4bb] Off topic - gene toxic for expression strains
Hi there, Somehow I've missed the original email :) Sorry! There are options for expressing really toxic genes, some of which have already been mentioned and others perhaps not: 1. tight regulation of expression (promoter, repressor, other regulatory elements, or a combination thereof). Beyond using araBAD, xylose promoter, rhamnose promoter, etc. there is one more thing that can be done, which is quite old school: phage induction. Yes, it means what it says - you add actual phage to the otherwise normal E.coli and the phage brings polymerase. This way there is no tangible expression except leakage which for T7 is close to non-existent. 2. change expression to a different organism: Bacillus, Pseudomonas or insect/mammalian cells 3. counter-expression of antisense RNA from a weaker promoter: this method is not exactly easy, but it does work -- you need a weak promoter (e.g. unmodified lac or trp) that would drive expression of antisense RNA. Easiest way to go about this is to put the antisense promoter on the 3' end of the gene, facing 'backwards'. Then, during induction, the sense-strand promoter is much more active and the sense RNA will win over the antisense. Good luck! Artem P.S. if you use 2% glucose in the media be prepared to fight acidification and the accumulation of Acetate in the spent medium, both of which can be a problem - sometimes severe - for 'weaker' E. coli strains. Strongly buffered medium is a must. - Cosmic Cats approve of this message On Mon, Apr 4, 2022 at 9:16 AM Nikolay Dobrev < nikolay.dob...@embl-hamburg.de> wrote: > 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 | facebook.com/embl.org | > youtube.com/user/emblmedia > Visit www.embl.org/events for a complete list of all EMBL events. > > > On 04/04/2022 10:32 AM Christian Roth 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 > 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 > 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/
Re: [ccp4bb] Off topic - gene toxic for expression strains
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 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 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/
Re: [ccp4bb] Off topic - gene toxic for expression strains
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 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 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/
[ccp4bb] Off topic - gene toxic for expression strains
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 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/
