demelerb@bionette.cgrb.orst.edu (Borries Demeler - Biophysics) (06/22/91)
Thanks to all who have replied to my request for help with rec- hosts. Since several people asked me to post a compilation of the replies, here they are: From jmasters@magnus.acs.ohio-state.edu Wed Jun 12 15:52:35 1991 Return-Path: <jmasters@magnus.acs.ohio-state.edu> Received: from top.magnus.acs.ohio-state.edu by cgrb.orst.edu (4.1/SMI-4.1) id AA15925; Wed, 12 Jun 91 15:52:32 PDT Received: by top.magnus.acs.ohio-state.edu (5.61/3.910213) id AA29919; Wed, 12 Jun 91 18:52:14 -0400 Date: Wed, 12 Jun 91 18:52:14 -0400 From: Jeffrey N Masters <jmasters@magnus.acs.ohio-state.edu> Message-Id: <9106122252.AA29919@top.magnus.acs.ohio-state.edu> To: demelerb@cgrb.orst.edu Subject: Re: E.coli host cells (rec deficient) Newsgroups: bionet.molbio.methds-reagnts In-Reply-To: <1991Jun12.192034.6348@lynx.CS.ORST.EDU> Organization: The Ohio State University Cc: Status: RO You might consider a stratagene bug, SRB or SRB(P2). They have deleted alot including DNA repair and claim that the bug is the next best thing to sliced bread. Because of the DNA repair mutation, the bug should be streaked out weekly from glycerol stock, and the plate wraped in foil to protect from light. An added advantage is kan resistance. sbcC, recJ, umuC::Tn5(KanR), uvrC, thi-1, endA1, supE44 (lac), gyrA96, relA1, mcrA, delta(mrr, hsdRMS, mcrBC) (F', proAB, lacIqZdeltaM15) The P2 dirivative helps in cloning methylated DNA. Good Luck Jeff Masters From prasher@hyatt.whoi.edu Thu Jun 13 04:54:45 1991 Return-Path: <prasher@hyatt.whoi.edu> Received: from aqua.whoi.edu by cgrb.orst.edu (4.1/SMI-4.1) id AA18996; Thu, 13 Jun 91 04:54:42 PDT Received: by aqua.whoi.edu (5.57/Ultrix2.4-C) id AA29376; Thu, 13 Jun 91 07:49:10 EDT Received: from hyatt.whoi.edu by hoh.mbl.edu. (4.1/SMI-4.1) id AA07835; Thu, 13 Jun 91 07:46:46 EDT Received: by hyatt.whoi.edu (4.1/SMI-4.1) id AA02078; Thu, 13 Jun 91 07:53:00 EDT Date: Thu, 13 Jun 91 07:53:00 EDT From: prasher@hyatt.whoi.edu (Doug Prasher) Message-Id: <9106131153.AA02078@hyatt.whoi.edu> To: demelerb@cgrb.orst.edu Subject: Re: E.coli host cells (rec deficient) Status: RO Borries: >Any suggestions of potentially useful hostcells would be appreciated. Have you tried a recBC strain? I can provide CES200, JC7623. So long, farewell. ******************************************************************** Douglas Prasher * prasher@hyatt.whoi.edu Woods Hole Oceanographic Institution * 508-457-2000 ext 2311 Biology Department * FAX: 508-457-2195 Woods Hole, MA 02543 * ******************************************************************** From bwoodman@magnus.acs.ohio-state.edu Thu Jun 13 09:55:55 1991 Return-Path: <bwoodman@magnus.acs.ohio-state.edu> Received: from bottom.magnus.acs.ohio-state.edu by cgrb.orst.edu (4.1/SMI-4.1) id AA20508; Thu, 13 Jun 91 09:55:52 PDT Received: by bottom.magnus.acs.ohio-state.edu (5.61/3.910213) id AA27607; Thu, 13 Jun 91 12:55:45 -0400 Date: Thu, 13 Jun 91 12:55:45 -0400 From: Robert H Woodman <bwoodman@magnus.acs.ohio-state.edu> Message-Id: <9106131655.AA27607@bottom.magnus.acs.ohio-state.edu> To: demelerb@cgrb.orst.edu Subject: Re: E.coli host cells (rec deficient) Newsgroups: bionet.molbio.methds-reagnts In-Reply-To: <1991Jun12.192034.6348@lynx.CS.ORST.EDU> Organization: The Ohio State University Status: RO In article <1991Jun12.192034.6348@lynx.CS.ORST.EDU> you write: >We are looking for an E. coli hostcell which prevents recombination >events in plasmids containing many repeats of the same gene. We tried >several recA- strains, but recombination still occured. Any suggestions >of potentially useful hostcells would be appreciated. >Please e-mail your help to >demelerb@bionette.cgrb.orst.edu >Thank you very much, -Borries Borries, Did you try DH5alpha and DH5alpha MCR from BRL? Did you try GM48 from IBI? If you tried those and still got recombination, then I can't help you. Bob Woodman -- ********************************************************************* *Robert H. "Bob" Woodman, PhD * "A job not worth doing well is not * *INTERNET: woodman.1@osu.edu * worth doing."--Salvador Luria * ********************************************************************* From brutlag@cmgm.Stanford.EDU Wed Jun 12 22:19:42 1991 Return-Path: <brutlag@cmgm.Stanford.EDU> Received: from cmgm.Stanford.EDU by cgrb.orst.edu (4.1/SMI-4.1) id AA17743; Wed, 12 Jun 91 22:19:35 PDT Received: by cmgm.Stanford.EDU (4.1/1.34) id AA08142; Wed, 12 Jun 91 22:19:29 PDT Date: Wed, 12 Jun 91 22:19:29 PDT From: brutlag@cmgm.Stanford.EDU (Douglas Brutlag) Message-Id: <9106130519.AA08142@cmgm.Stanford.EDU> To: demelerb@cgrb.orst.edu Subject: Re: E.coli host cells (rec deficient) Newsgroups: bionet.molbio.methds-reagnts References: <1991Jun12.192034.6348@lynx.CS.ORST.EDU> Status: RO Borries, You may be out of luck. I ran into this same problem attempting to clone tandemly repeated satellite DNAs into bacterial plasmids. We examined many recombination deficient strains all to no avail. The only solution we found that stabilized the repeats was to have the total length of the tandemly repeated region shorter than an average okazaki fragment. We felt that the recombination was caused by unequal exchange during replication. Here are two references and their abstracts: Brutlag, D., Fry, K., Nelson, T. and Hung, P. (1977). Synthesis of hybrid bacterial plasmids containing highly repeated satellite DNA.. Cell, 10(3), 509-19. Hybrid plasmid molecules containing tandemly repeated Drosophila satellite DNA were constructed using a modification of the (dA)-(dT) homopolymer procedure of Lobban and Kaiser (1973). Recombinant plasmids recovered after transformation of recA bacteria contained 10% of the amount of satellite DNA present in the transforming molecules. The cloned plasmids were not homogenous in size. Recombinant plasmids isolated from a single colony contained populations of circular molecules which varied both in the length of the satellite region and in the poly(dA)-(dt) regions linking satellite and vector. While subcloning reduced the heterogeneity of these plasmid populations, continued cell growth caused further variations in the size of the repeated regions. Two different simple sequence satellites of Drosophila melanogaster (1.672 and 1.705 g/cm3) were unstable in both recA and recBC hosts and in both pSC101 and pCR1 vectors. We propose that this recA-independent instability of tandemly repeated sequences is due to unequal intramolecular recombination events in replicating DNA molecules, a mechanism analogous to sister chromatid exchange in eucaryotes. Lohe, A. R. and Brutlag, D. L. (1986). Multiplicity of satellite DNA sequences in Drosophila melanogaster.. Proc Natl Acad Sci U S A, 83(3), 696-700. Three Drosophila melanogaster satellite DNAs (1.672, 1.686, and 1.705 g/ml in CsCl), each containing a simple sequence repeated in tandem, were cloned in pBR322 as small fragments about 500 base pairs long. This precaution minimized deletions, since inserts of the same size as the fragments used for cloning were recovered in a stable form. A homogeneous tandem array of one sequence type usually extended the length of the insert. Eleven distinct repeat sequences were discovered, but only one sequence was predominant in each satellite preparation. The remaining classes were minor in amount. The repeat unit lengths were restricted to 5, 7, or 10 base pairs, with sequences closely related. Each sequence conforms to the expression (RRN)m(RN)n, where R is A or G. The multiplicity of simple repeated sequences revealed despite the small sample size suggests that numerous repeat sequences reside in heterochromatin and that particular rules apply to the structure of the repeating sequence. We also tried host mutants that altered the size of Okazaki fragments and these had deleterious effects on the stability of the entire plasmid. Sorry. Doug Brutlag From brutlag@cmgm.Stanford.EDU Thu Jun 13 11:35:57 1991 Return-Path: <brutlag@cmgm.Stanford.EDU> Received: from cmgm.Stanford.EDU by cgrb.orst.edu (4.1/SMI-4.1) id AA21312; Thu, 13 Jun 91 11:35:51 PDT Received: from (Transcript.Stanford.EDU) by cmgm.Stanford.EDU (4.1/1.34) id AA09159; Thu, 13 Jun 91 11:35:35 PDT Date: Thu, 13 Jun 1991 11:37:13 PDT From: Doug Brutlag <brutlag@cmgm.Stanford.EDU> Subject: Re: E.coli host cells (rec deficient) To: demelerb@cgrb.orst.edu In-Reply-To: Your message of Thu, 13 Jun 91 10:08:55 PDT Message-Id: <MacMS.2409.16838.brutlag@cmgm.stanford.edu> Status: R Borries, We found that a longer DNA repeat was stable in recA- cells (a 359 base pair repeat and a 254 bp repeat) but we weren't sure whether this stability was due to the nature or the length of the repeating unit. We did do an experiment growing the cells with 16 tandem repeats of 359 base pairs for over 150 generations with repeated serial dilutions of the culture and found only a single major deletion event late in the growth. The references for you to look at with respect to stability of longer repeats (254 and 259 bp) are: Carlson, M. and Brutlag, D. (1977). Cloning and characterization of a complex satellite DNA from Drosophila melanogaster. From 21337MGR@msu.edu Thu Jun 13 18:07:27 1991 Return-Path: <21337MGR@msu.edu> Received: from msu.edu (ibm.cl.msu.edu) by cgrb.orst.edu (4.1/SMI-4.1) id AA24215; Thu, 13 Jun 91 18:07:23 PDT Message-Id: <9106140107.AA24215@cgrb.orst.edu> Received: from MSU.BITNET by msu.edu (IBM VM SMTP R1.2.2MX) with BSMTP id 5945; Thu, 13 Jun 91 21:08:11 EDT Received: by MSU (Mailer R2.03B) id 5943; Thu, 13 Jun 91 21:08:10 EDT Date: Thursday, 13 June 1991 9:07pm ET To: demelerb@cgrb.orst.edu From: "Jonathan.Walton" <21337MGR@msu.edu> Subject: E.coli host cells (rec deficient Status: R I've heard that SURE cells from Stratagene are excellent for propagating "difficult" DNA. I am about to use them to make a cosmid library in which the DNA we are interested in has a lot of repeated DNA. J. Walton (21337mgr@msu.bitnet) Michigan State University From TAN@CUMULI.vmsmail.ethz.ch Fri Jun 14 02:07:06 1991 Return-Path: <TAN@CUMULI.vmsmail.ethz.ch> Received: from bernina.ethz.ch by cgrb.orst.edu (4.1/SMI-4.1) id AA26289; Fri, 14 Jun 91 02:07:03 PDT Message-Id: <9106140907.AA26289@cgrb.orst.edu> Received: from CUMULI by bernina.ethz.ch id <5120-0@bernina.ethz.ch>; Fri, 14 Jun 1991 10:57:35 +0200 Received: from CUMULI.vmsmail.ethz.ch by ETHZ.vmsmail.ethz.ch with MAIL-11 (pp-mail11d 0.0); Fri, 14 Jun 91 10:57:35 MET DST Date: Fri, 14 Jun 91 10:57:35 MET DST From: TAN@CUMULI.vmsmail.ethz.ch X-Mail11-To: ETHZ::"demelerb@bionette.cgrb.orst.edu" Subject: recA- strain To: demelerb@cgrb.orst.edu Status: R Dear Borries, Stratagene sells their SURE strain which is uvrC umuC sbcC recJ recB endA ... . We have used it as the host for plasmids that contain multiple repeats and have found it to be better than HB101 for maintaining these plasmids. Recombination still occurs, but less frequently. On the downside, the SURE strain grows very slowly. If you hear about other recombination deficient strains that are effective in maintaining plasmids with multiple repeats, I would be very interested to learn about them. Best regards. Song Tan Institute for Molecular Biology and Biophysics ETH - Honggerberg 8093 Zurich Switzerland email: TAN@CUMULI.VMSMAIL.ETHZ.CH From @CUNYVM.CUNY.EDU:CONTRERA@MOLEBIOL.RUG.AC.BE Fri Jun 14 04:06:49 1991 Return-Path: <@CUNYVM.CUNY.EDU:CONTRERA@MOLEBIOL.RUG.AC.BE> Received: from CUNYVM.CUNY.EDU by cgrb.orst.edu (4.1/SMI-4.1) id AA26520; Fri, 14 Jun 91 04:06:44 PDT Received: from BGERUG51.BITNET by CUNYVM.CUNY.EDU (IBM VM SMTP R1.2.2MX) with BSMTP id 7512; Fri, 14 Jun 91 07:06:30 EDT Received: from CDR.RUG.AC.BE (UNKNOWN@UNKNOWN) by BGERUG51.BITNET with PMDF#10528; Fri, 14 Jun 1991 13:05 N Date: Fri, 14 Jun 91 13:04 N From: CONTRERA%MOLEBIOL.RUG.AC.BE@CUNYVM.CUNY.EDU Subject: Borries:recombination negative coli To: demelerb@cgrb.orst.edu Message-Id: <D0F77FE280003D57@BGERUG51.BITNET> X-Envelope-To: demelerb@bionette.cgrb.orst.edu Status: R We have used the SURE strain from Stratagene with succes to construct a trimer of human TNF (tumor necroasis factor). In my experience this is the best strain for such purposes. Best regards R. Contreras From AHARRIS@AC.DAL.CA Sun Jun 16 18:36:30 1991 Return-Path: <AHARRIS@AC.DAL.CA> Received: from AC.DAL.CA by cgrb.orst.edu (4.1/SMI-4.1) id AA00961; Sun, 16 Jun 91 18:36:26 PDT Date: Sun, 16 Jun 91 22:28 -0300 From: AHARRIS@AC.DAL.CA Subject: E.coli host strains To: demelerb@cgrb.orst.edu Message-Id: <5C4E249C9543001BCE@AC.DAL.CA> X-Envelope-To: demelerb@bionette.cgrb.orst.edu X-Vms-To: IN%"demelerb@bionette.cgrb.orst.edu" Status: R In regard to your posting on bionet, have you tried Stratagene "Sure" cells? They are claimed to be Rec BC defficient. We have used them to try to clone minisatellite sequences (unfortunately with little success) in an attempt to stop the arrays from collapsing to a couple of repeat units. We have yet to find a host strain that prevents deletions in these sequences but "Sure" cells may work for you. I would be grateful to hear if you come up with any alternative hosts. Good luck. Andrew Harris Marine Gene Probe Lab Dalhousie University Halifax, Nova Scotia, Canada From macbeth@opal.cs.tu-berlin.de Mon Jun 17 16:17:59 1991 Return-Path: <macbeth@opal.cs.tu-berlin.de> Received: from opal.cs.tu-berlin.de by cgrb.orst.edu (4.1/SMI-4.1) id AA07792; Mon, 17 Jun 91 16:17:47 PDT Received: from oppdal.cs.tu-berlin.de by opal.cs.tu-berlin.de with SMTP id AA20874 (5.65a/IDA-1.4.2 for demelerb@CGRB.ORST.EDU); Tue, 18 Jun 91 01:17:01 +0200 Date: Tue, 18 Jun 91 01:17:01 +0200 From: Andreas Pahl <macbeth@opal.cs.tu-berlin.de> Message-Id: <9106172317.AA20874@opal.cs.tu-berlin.de> To: demelerb@cgrb.orst.edu (Borries Demeler - Biochem) Subject: Re: E.coli host cells (rec deficient) Status: RO In article <1991Jun12.192034.6348@lynx.CS.ORST.EDU> you write: > We are looking for an E. coli hostcell which prevents recombination > events in plasmids containing many repeats of the same gene. We tried > several recA- strains, but recombination still occured. Any suggestions > of potentially useful hostcells would be appreciated. > Please e-mail your help to > demelerb@bionette.cgrb.orst.edu > > Thank you very much, -Borries -- Hi, did you try Stratagenes E.coli SURE (Stop unwanted Rearrangement events or something like this written). The cells grow rather slowly, but you might try this one. Tschau, Andreas P.S. I suppose you tried normal strains like DH5alpha or so. ############################################################################### Andreas Pahl macbeth@opal.cs.tu-berlin.de Institut f. Biochemie u. Molekulare Biologie TU Berlin Franklinstr. 29 D-1000 Berlin 10 ############################################################################### From mytelka@enzyme.berkeley.edu Mon Jun 17 20:24:12 1991 Return-Path: <mytelka@enzyme.berkeley.edu> Received: from violet.berkeley.edu by cgrb.orst.edu (4.1/SMI-4.1) id AA09110; Mon, 17 Jun 91 20:24:05 PDT Received: from enzyme.Berkeley.EDU by violet.berkeley.edu (5.61/1.32) id AA05397; Mon, 17 Jun 91 20:23:55 PDT Received: from codon9.Berkeley.EDU by enzyme (4.0/SMI-3.0DEV3.7) id AA15460; Mon, 17 Jun 91 20:19:38 PDT Date: Mon, 17 Jun 91 20:19:38 PDT From: mytelka@enzyme.Berkeley.Edu (Daniel Mytelka) Message-Id: <9106180319.AA15460@enzyme> To: demelerb@cgrb.orst.edu Subject: rec- Status: RO I would try talking to the people at Gerry Smith's lab at the Hutch in Seattle. I got a good recA-BC- strain from them a few years back (when I was in a different lab). -Dan Mytelka