xl1-blue is not an expression host,since i have cloned it
successfully,i need to transform into expression host, i am able to
transform it into dh5 alfa,but not in any of expression host
------------------------------------------------------------------------
*From:* ar...@xtals.org [mailto:ar...@xtals.org]
*Sent:* Mon 5/4/2009 6:32 PM
*To:* atul kumar
*Cc:* ccp4bb@jiscmail.ac.uk
*Subject:* Re: [ccp4bb] problem in transformation of pqe 30 clone
Hi,
You are using a pQE vector which has a T5 promoter. T5 is a native-like
promoter, recognized by the E. coli RNA polymerase - and this means that
even with lac operatorsupstream there is a huge amount of leakage in this
system. If your protein is even moderately toxic then you have issues.
Your solutions are
1) to use cells with higher levels of lac repressor (XL1-blue for example)
2) to re-clone this ORF under some tightly controlled promoter
Artem
>
> i have cloned my gene successfully into qiagen vector into pqe30 but
i do
> transformation of this into BL21,pLys,Rosseta,C41, i dont get any
> colonies,comp cells are good other clones give good no of colonies upon
> transformation.
> can someone help???
> thanks
> atul
>
> -----Original Message-----
> From: CCP4 bulletin board on behalf of
herman.schreu...@sanofi-aventis.com
> Sent: Mon 5/4/2009 3:49 PM
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: Re: [ccp4bb] Peptide on two-fold axis - was:[ccp4bb] PEG
molecule
> crossing a two-fold crystallographic symmetry axis
>
> Dear Lidefeng,
>
> One "Tassos" afterthought; you are sure you traced the chain
correctly? It
> is not that both change make a crystal contact and then each go
their own
> (disordered) way?
>
> Best regards,
> Herman
>
> -----Original Message-----
> From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of
> lidefeng
> Sent: Friday, May 01, 2009 2:48 PM
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: Re: [ccp4bb] Peptide on two-fold axis - was:[ccp4bb] PEG
molecule
> crossing a two-fold crystallographic symmetry axis
>
> Dear Herman.Schreuder,
>
> Perhaps there is some misunderstanding about my question.
There is
> one molecule in the asymmetric unit (showed as symbos A). After the
> 2nd crystallographic symmetric operation, another molecule appears
> (symbol B). However, the density show that there is one peptide chain
> cross the 2nd axis (symbol C). If we give chain C occupancy of 0.5,
> only 50% of chain C belongs to molecules A and B, respectively. In
> another word, , it looks like one half of chain C belongs to molecule
> A and the other belongs to B. It means that one half of chain C
> belongs to molecule A disappear, so do that belongs to molecule B.
>
> 2nd axis
> |
> AAAAAA | BBBBBB
> AAAAAA | BBBBBB
> AA | BB
> \ | /
> CCCCCCC|CCCCCCC |
> |
> |
> Your sincerely
> ????????De-Feng Li
> ????????lidef...@moon.ibp.ac.cn
> ??????????2009-05-01
>
> Defeng Li, Dr.,
> Email: lidef...@moon.ibp.ac.cn
> National Laboratory of Biomacromolecules, Institute of Biophysics,
Chinese
> Academy of Sciences,
> 15 Datun Road, Chaoyang District,
> Beijing 100101, China
>
>
> ======= 2009-04-30 14:52:00 You writed in your letter:=======
>
>>Dear Feng-Li,
>>
>>The other half occupancy peptide is generated by crystallographic
>> symmetry (the twofold), you need only to build one. To check that
>> everything fits properly, you should switch on the crystallographic
>> symmetry in coot. (Draw -> Cell & Symmetry).
>>
>>Good luck!
>>Herman
>>
>>
>>-----Original Message-----
>>From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of
>>lidefeng
>>Sent: Thursday, April 30, 2009 3:44 AM
>>To: CCP4BB@JISCMAIL.AC.UK
>>Subject: Re: [ccp4bb] Peptide on two-fold axis - was:[ccp4bb] PEG
>>molecule crossing a two-fold crystallographic symmetry axis
>>
>>Dear Tim Gruene,
>>
>> But how to illustrate the other one half occupancy of peptide?
>> Disorder ?
>>
>>
>> Your sincerely
>>????????De-Feng Li
>>????????lidef...@moon.ibp.ac.cn
>>??????????2009-04-30
>>
>>Defeng Li, Dr.,
>>Email: lidef...@moon.ibp.ac.cn
>>National Laboratory of Biomacromolecules, Institute of Biophysics,
>>Chinese Academy of Sciences,
>>15 Datun Road, Chaoyang District,
>>Beijing 100101, China
>>
>>
>>======= 2009-04-29 10:55:00 You writed in your letter:=======
>>
>>>Hello De-Feng Li,
>>>
>>>first of all sorry for changing the subject: I think starting a new
>>>thread from an existing one may hamper people who are going to search
>>>the archives in the future, therefore it is good practice to give it
>>>its separate subject line, even though it certainly is be very closely
>>>related.
>>>
>>>In your case you can refine two peptides each with an occupancy of
>>>0.5, one for each direction.
>>>
>>>Tim
>>>--
>>>Tim Gruene
>>>Institut fuer anorganische Chemie
>>>Tammannstr. 4
>>>D-37077 Goettingen
>>>
>>>GPG Key ID = A46BEE1A
>>>
>>>
>>>On Wed, 29 Apr 2009, lidefeng wrote:
>>>
>>>> Hi everyone,
>>>>
>>>> Following Chandrika's question, what should I do if one peptide
>>>> chain crosses a two-fold crystallographic symmetry axis?
>>>> The peptide is not symmetric and the sidechain of one Se-Met (two
>>>> after CS operation) is determined and conformed by MAD.
>>>>
>>>> Your sincerely
>>>> ????????De-Feng Li
>>>> ????????lidef...@moon.ibp.ac.cn
>>>> ??????????2009-04-29
>>>>
>>>> Defeng Li, Dr.,
>>>> Email: lidef...@moon.ibp.ac.cn
>>>> National Laboratory of Biomacromolecules, Institute of Biophysics,
>>>> Chinese Academy of Sciences,
>>>> 15 Datun Road, Chaoyang District,
>>>> Beijing 100101, China
>>>>
>>>>
>>>> ======= 2009-04-29 17:02:00 You writed in your letter?=======
>>>>
>>>>> Hello everyone,
>>>>>
>>>>> My protein crystallised in the spacegroup P6522 with one protein
>>>>> molecule in the asymmetric unit. I have a PEG molecule from the
>>>>> crystallization condition which crosses a two-fold crystallographic
>>>>> symmetry axis. PEG is symmetric hence this does not violate the
>>>>> crystal symmetry. However, this situation causes two problems
which I
>>>>> need to solve :
>>>>>
>>>>> First, How can I refine this structure ? I am using Phenix. Is there
>>>>> a way to remove van der Waals repulsion between one half occupancy
>>>>> PEG and its crystallographic symmetry mate ?
>>>>>
>>>>> Second, how do I submit this structure to PDB ? Do I include a full
>>>>> PEG molecule at half occupancy even though one half is related
to the
>>>>> other via crystallographic symmetry ?
>>>>>
>>>>> Thanks,
>>>>> Chandrika
>>>>
>>>> ========================================================
>>>>
>>>>
>>
>>========================================================
>>
>
> ========================================================
>
>
>
