Re: [ccp4bb] Off topic - transformation problems
You may also want to try 0.5 - 2% (w/v) glucose in your plates alongside transformation into the NEB T7 Express strains: http://www.neb.com/nebecomm/products/productC3013.asp http://www.neb.com/nebecomm/products/faqproductC3013.asp LysY gives you higher final ODs [no lysozyme activity] than using pLysS/E and is compatible with the T7 promoter in your pET20b. Renos On 14/07/2011 03:33, Dima Klenchin wrote: In this case, pGEX4T3 vector also expressed inserts constitutively. http://www.biovisualtech.com/bvplasmid/pGEX-4T-3.htmhttp://www.biovisualtech.com/bvplasmid/pGEX-4T-3.htm Not quite. pGEX vectors carry a copy of lacI^q, resulting in very low leak expression from PTAC promoter. Definitely lower than the relatively leaky T7 promoter in pET20, which does not express laqI^q. Switching to a plasmid from higher pET series that do have laqI^q (e.g. pET31) should reduce the leakiness. I also thought that target protein may have tocicity on host strain. But, why pGEX4T3-target protein was not show the same phenomena. Now, we are trying to change the host BL21(DE3) to Rosetta(DE3) and BL21(DE3)pLysS. pLysS should help with repression under non-inducing conditions. If it is true toxicity issue, you might need to switch to plates with synthetic medium that does not contain any traces of lactose. - Dima
Re: [ccp4bb] Off topic - transformation problems
On Thu, 14 Jul 2011, Wonjin Bae wrote: Hi, all Recently, some bactrial source enzyme(sialidase) was subcloned pET20b and pGEX4T3 vector. Then, these two construct were transformed to BL21(DE3) expression host. DNA sequencing results were accurate. In case of pGEX, many colony was formed and shown the high level of expression. But, colony was not shown in pET20b case. (no one colony) In case of mock-pET20a vector transformed very well. Simplest explanation - wrong antibiotic used on the pET20b case? What's the problems? I never experieced transfomation problems. Does anyone know how to solve the this problems? Thanks a lot !! Genie, Eric T. Larson, PhD Biomolecular Structure Center Department of Biochemistry Box 357742 University of Washington Seattle, WA 98195 email: larso...@u.washington.edu
Re: [ccp4bb] Off topic - transformation problems
oops - I just caught that the empty pET20 did transform well so the other suggestions about toxicity are probably more accurate. On Thu, 14 Jul 2011, Eric Larson wrote: On Thu, 14 Jul 2011, Wonjin Bae wrote: Hi, all Recently, some bactrial source enzyme(sialidase) was subcloned pET20b and pGEX4T3 vector. Then, these two construct were transformed to BL21(DE3) expression host. DNA sequencing results were accurate. In case of pGEX, many colony was formed and shown the high level of expression. But, colony was not shown in pET20b case. (no one colony) In case of mock-pET20a vector transformed very well. Simplest explanation - wrong antibiotic used on the pET20b case? What's the problems? I never experieced transfomation problems. Does anyone know how to solve the this problems? Thanks a lot !! Genie, Eric T. Larson, PhD Biomolecular Structure Center Department of Biochemistry Box 357742 University of Washington Seattle, WA 98195 email: larso...@u.washington.edu
[ccp4bb] Off topic - transformation problems
Hi, all Recently, some bactrial source enzyme(sialidase) was subcloned pET20b and pGEX4T3 vector. Then, these two construct were transformed to BL21(DE3) expression host. DNA sequencing results were accurate. In case of pGEX, many colony was formed and shown the high level of expression. But, colony was not shown in pET20b case. (no one colony) In case of mock-pET20a vector transformed very well. What's the problems? I never experieced transfomation problems. Does anyone know how to solve the this problems? Thanks a lot !! Genie,
Re: [ccp4bb] Off topic - transformation problems
I never used pET20b, but I found on the Internet that it expresses inserts constitutively. Since the cloned protease should be toxic to cells, its constitutive expression might prevent the formation of colonies. But there might be millions of other reasons. Why did you use pET20b? http://www.biovisualtech.com/bvplasmid/pET-20b%28+%29.htm Alex On Jul 13, 2011, at 5:54 PM, Wonjin Bae wrote: Hi, all Recently, some bactrial source enzyme(sialidase) was subcloned pET20b and pGEX4T3 vector. Then, these two construct were transformed to BL21(DE3) expression host. DNA sequencing results were accurate. In case of pGEX, many colony was formed and shown the high level of expression. But, colony was not shown in pET20b case. (no one colony) In case of mock-pET20a vector transformed very well. What's the problems? I never experieced transfomation problems. Does anyone know how to solve the this problems? Thanks a lot !! Genie,
Re: [ccp4bb] Off topic - transformation problems
Thank you for your kind advices. In this case, pGEX4T3 vector also expressed inserts constitutively. http://www.biovisualtech.com/bvplasmid/pGEX-4T-3.htm I also thought that target protein may have tocicity on host strain. But, why pGEX4T3-target protein was not show the same phenomena. Now, we are trying to change the host BL21(DE3) to Rosetta(DE3) and BL21(DE3)pLysS. Is this try may helpful to solve the this kind of problems? Thanks for your valuable comments. Genie 2011/7/14 aaleshin aales...@burnham.org I never used pET20b, but I found on the Internet that it expresses inserts constitutively. Since the cloned protease should be toxic to cells, its constitutive expression might prevent the formation of colonies. But there might be millions of other reasons. Why did you use pET20b? http://www.biovisualtech.com/bvplasmid/pET-20b%28+%29.htm Alex On Jul 13, 2011, at 5:54 PM, Wonjin Bae wrote: Hi, all Recently, some bactrial source enzyme(sialidase) was subcloned pET20b and pGEX4T3 vector. Then, these two construct were transformed to BL21(DE3) expression host. DNA sequencing results were accurate. In case of pGEX, many colony was formed and shown the high level of expression. But, colony was not shown in pET20b case. (no one colony) In case of mock-pET20a vector transformed very well. What's the problems? I never experieced transfomation problems. Does anyone know how to solve the this problems? Thanks a lot !! Genie, -- Jin Soo Bae 790-784 room 204, Dept of Life Science, POSTECH, san31, Hyoja-dong, Nam-gu, Pohang, Gyungbuk, Korea tel:82-54-279-8627 fax:82-54-279-8111
Re: [ccp4bb] Off topic - transformation problems
In this case, pGEX4T3 vector also expressed inserts constitutively. http://www.biovisualtech.com/bvplasmid/pGEX-4T-3.htmhttp://www.biovisualtech.com/bvplasmid/pGEX-4T-3.htm Not quite. pGEX vectors carry a copy of lacI^q, resulting in very low leak expression from PTAC promoter. Definitely lower than the relatively leaky T7 promoter in pET20, which does not express laqI^q. Switching to a plasmid from higher pET series that do have laqI^q (e.g. pET31) should reduce the leakiness. I also thought that target protein may have tocicity on host strain. But, why pGEX4T3-target protein was not show the same phenomena. Now, we are trying to change the host BL21(DE3) to Rosetta(DE3) and BL21(DE3)pLysS. pLysS should help with repression under non-inducing conditions. If it is true toxicity issue, you might need to switch to plates with synthetic medium that does not contain any traces of lactose. - Dima
