From skuehn from mail.rockefeller.edu Mon Jan 5 15:03:55 2009 From: skuehn from mail.rockefeller.edu (Seppe Kuehn) Date: Mon Jan 5 22:55:11 2009 Subject: [Chlamydomonas] how to limit growth? Message-ID: We are looking for a protocol for growing Chlamydomonas Reinhardtii under phototrophic conditions where the final cell density is limited to ~10^5 cells/mL. We would like to do this without adversely effecting motility, metabolism, or overall health of the culture. We are currently working with the UTEX 2244 (mt+) strain (also known as CC-125) but are open to using other strains if necessary. Thus far Sager's medium has worked best for our application so a simple modification to this medium that limits final cell density would be ideal. Thank you! From dactyl from ll.mit.edu Tue Jan 6 16:28:48 2009 From: dactyl from ll.mit.edu (Boettcher, Tara) Date: Tue Jan 6 17:51:25 2009 Subject: [Chlamydomonas] Transformation Message-ID: I am using CC-125 and CC-124 and trying to transform them with pSP124S with Bleo resistance marker, using both Karen Kindle method with glass beads and the Purton variation of the Karen Kindle method, and not having any luck. I've done an agar kill curve with zeocin, I have linearized the plasmid, and have tried autolysin treatments. Does anybody have insight on 'crucial steps', a new method to try, or a new plasmid with a different resistance marker I could try? Any help is greatly appreciated! From jebsen from rz.uni-leipzig.de Wed Jan 7 04:38:15 2009 From: jebsen from rz.uni-leipzig.de (Christian Jebsen) Date: Wed Jan 7 07:13:49 2009 Subject: [Chlamydomonas] how to limit growth In-Reply-To: <200901061704.n06H4Uj01003@net.bio.net> References: <200901061704.n06H4Uj01003@net.bio.net> Message-ID: <49647807.9050002@rz.uni-leipzig.de> A turbidostat will be the right culturing method for you, this would be a technical way to limit the cell density. A change of the culture media or rather the nutrient supply to limit cell density would always effect the motility and the health status of the cells. The cells could loose their motility and would reduce their metabolic turnover. chlamy-request@oat.bio.indiana.edu wrote: > Send Chlamy mailing list submissions to > chlamy@net.bio.net > > To subscribe or unsubscribe via the World Wide Web, visit > http://www.bio.net/biomail/listinfo/chlamy > or, via email, send a message with subject or body 'help' to > chlamy-request@net.bio.net > > You can reach the person managing the list at > chlamy-owner@net.bio.net > > When replying, please edit your Subject line so it is more specific > than "Re: Contents of Chlamy digest..." > > > Today's Topics: > > 1. how to limit growth? (Seppe Kuehn) > > > ---------------------------------------------------------------------- > > Message: 1 > Date: Mon, 5 Jan 2009 15:03:55 -0500 > From: Seppe Kuehn > Subject: [Chlamydomonas] how to limit growth? > To: chlamy@magpie.bio.indiana.edu > Message-ID: > > Content-Type: text/plain; charset=US-ASCII; delsp=yes; format=flowed > > We are looking for a protocol for growing Chlamydomonas Reinhardtii > under phototrophic conditions where the final cell density is limited > to ~10^5 cells/mL. We would like to do this without adversely > effecting motility, metabolism, or overall health of the culture. > We are currently working with the UTEX 2244 (mt+) strain (also known > as CC-125) but are open to using other strains if necessary. Thus > far Sager's medium has worked best for our application so a simple > modification to this medium that limits final cell density would be > ideal. Thank you! > > ------------------------------ > > _______________________________________________ > Chlamy mailing list > Chlamy@net.bio.net > http://www.bio.net/biomail/listinfo/chlamy > > End of Chlamy Digest, Vol 40, Issue 1 > ************************************* > -- Christian Jebsen (Dipl.-Biol.) University of Leipzig Department of Plant Physiology Johannisallee 21, 04103 Leipzig Phone: #49-341-9736876 Fax:#49-341-9736899 jebsen@rz.uni-leipzig.de, http://www.uni-leipzig.de/~pflaphys From jonathanmeuser from gmail.com Thu Jan 8 12:28:23 2009 From: jonathanmeuser from gmail.com (jonathanmeuser@gmail.com) Date: Thu Jan 8 13:13:44 2009 Subject: [Chlamydomonas] Re: Transformation References: Message-ID: <59ad1f17-f788-484c-83c0-4e98db80e512@c36g2000prc.googlegroups.com> On Jan 6, 2:28?pm, "Boettcher, Tara" wrote: > I am using CC-125 and CC-124 and trying to transform them with pSP124S with Bleo resistance marker, using both Karen Kindle method with glass beads and the Purton variation of the Karen Kindle method, and not having any luck. > I've done an agar kill curve with zeocin, I have linearized the plasmid, and have tried autolysin treatments. > > Does anybody have insight on 'crucial steps', a new method to try, or a new plasmid with a different resistance marker I could try? > Any help is greatly appreciated! Dear Tara, I have been transforming Chlamydomonas D66 with the sSP124S plasmid because my background mutant already has the Streptomyces rimosus aphVII (pSI103 plasmid with a aminoglycoside phosphotransferase conferring paromomycin resistance) marker (Sizova et al. 2001). You can readily get this plasmid from the Chlamy Center (http:// www.chlamy.org/plasmids.html) and it seems to be a marker that is much easier to use. As I understand, the BleR marker has fallen out of favor because of some of the difficulties that can occur. For one, there seems to be different strengths of Bleocin/Zeocin from batch to batch and each order should be titrated separately. Further, the Sizova paper mentions that the BLE protein dimer can only inactivate two antibiotic molecules through drug sequestration. So it is as if the BLE resistance is quenching the drug. Thus, variable expression levels in your mutants should confer a pool of variable resistance in the population of mutants you plate. If your concentration of Ble is too high, you will select for mutants with mulitiple resistance gene insertions. I have also noticed variable susceptibility to the zeocin antibiotic in the wild-type cells based on factors like culture growth stage and, of course, the density of cells plated. When working with Ble-resistance it is important to have a good negative control that goes through the entire transformation process, but without the introduction of the plasmid. This is important, because the current electroporation method I am employing makes even the wild-type cells more susceptible to bleocin. I suspect, but have not tested, that this is due to a combination of diluted cell number during transfers and cell mortality during electroporation. Zeocin is also a mutagen, which can result in point mutations unrelated to your site of insertion, making tracking down the cause of a mutant phenotype nearly impossible. Microscopy confirms that the bead transformation methods lyses many cells. As with your experience, I have had poor transformation efficiency with the bead method, but it does work. It is important to try lots of variations and keep records of everything (including cell density at start of experiment ect.). I have also had good luck with increasing the the plasmid concentration to make the bead beating method work, but this has been reported to also increase the number of transformants with multiple insertions. You can also try plating the cells with starch. Overall, you will likely have the best luck with the pSI103 plasmid and electroporation. The APH protein inactivates paromomycin by transferring a phosphate from ATP to the paromomycin antibiotic, and can keep catalyzing this reaction. Thus, the effective range of paromomycin, paromomycin:cell ratio, and variability due to variability in gene expression/gene copy number reasoned to be much better. In my hands, this seems to be the case. Hope this helps! -Jonathan Meuser From david.dauvillee from univ-lille1.fr Mon Jan 12 05:27:55 2009 From: david.dauvillee from univ-lille1.fr (=?ISO-8859-1?Q?David_Dauvill=E9e?=) Date: Tue Jan 13 10:49:31 2009 Subject: [Chlamydomonas] Chlamydomonas electroporation with Bio-Rad GenePulser Xcell Message-ID: <496B1B2B.7040700@univ-lille1.fr> Hi, does anybody know a good setting on the Bio-Rad GenePulser Xcell apparatus to electroporate Chlamy? We were used to electroporate the algae with the Eppendorf Multiporator in prokaryotic mode at 750V and 5ms but our numerous attempts to reproduce that on the new apparatus failed. We are using the sucrose/starch protocol established to electroporate a cw15 strain. Thank you David -- David Dauvill?e *"Biochemical Genetics and Functional Biology"* Unit? de Glycobiologie Structurale et Fonctionnelle UMR 8576 CNRS USTL / B?t C9 59650 Villeneuve d'Ascq FRANCE Tel: +33 3 20 43 65 43 Fax: +33 3 20 43 65 55 David.Dauvillee@univ-lille1.fr