Learned something new about chlorella cell walls. Thanks a bunch! :)
I would say that nuclear is easier if you get agro to freak out via acetosyringone or vanilla extract. Chloroplast requires some thought for homogous integration vectors...or just get one ready-made from a university lab. Thing is the algal cell needs to be wounded a bit for agro...which is hard to do in the case of small single cells. Maybe a co-culture of agro, chlorella, and glass beads. A quick vortex and then let it sit for 15m or so? Then plate on timentin or other penecillin base with beta lactamase inhibitors?
If you want a quick and easy method, one of which I, along with Antonio Lamb, will be trying out personally in the days and weeks to come, why not try a cell wall deficient chlamydonomas strain like CC-400 and use glass beads and plasmid? You can follow our progress at experiment.com under the algae oral vaccine insulin project. Lots of pics soon. Just got the strain yesterday!
--hey, yea i just need to get the plasmid inside and take pictures of the reporter( luciferin ) and i am done here. I have heard ( not read all the papers yet) that expression in chloroplast is easier to produce but im not sure I ''assume'' with electroporation it will go to Chlorop.. and not into nucleous. The chlorella's genome is if im not mistaken 38M bp chloroplast arund 150k and mtDNA around 55k.As far as i have found from papers cell walls vary alot in algae, Nannochloropsis as i ve read has Alganan cellulose and lignin but chlorella's vary alot, the vulgaris doesnt have cellulose at all but it has inner matrix and outter layered N-acetylglucosomine and another mannose based sugar. For the cell wall degradation it would require at least 3 enzymes ( from one of the protocols) chitinanase chitinase and chitosanase which cost more then they should in my case lol so i would rather go wtih the electroporation or sonication protocol. But i want to try salt first to see if there will be atleast any effect since it is fairly cheap and fast method.But yea so far I am just trying to work on the protocol and different promoters to establish the solid and very cheap protocol.
On Wednesday, December 16, 2015 at 11:08:07 AM UTC-8, Sebastian wrote:Are you trying to do transient or stable transformation? Nuclear or chloroplast integration? Is the genome of nucleus and chloroplast known for both species?IIRC chlorella has a cell wall which prevents efficient ingress of just about anything. Protoplasting via cellulase may increase CaCl2 but I've read a few papers on chlorella a while back citing electroporation being the "bee's knees" but heck, electroporation can insert DNA into most living tissues so it should be a surefire way. If you do go the protoplast route, give polyethylene glycol (PEG) a chance. Its more gentle and less salty. Either way for transient expression you just need to get the plasmid in there, for actual stable integration...thats a whole different ball game.test--On Wed, Dec 16, 2015 at 10:46 AM, Alex D <alexx...@gmail.com> wrote:Hello everyone,--I am currently working on a project which requires transfection of Chlorella Vulgaris and nannochloropsis. Plasmid size is approx, 6K bp, but I am more concerned about the most efficient and cost effective technique for transfection. Initial experiment will be carried with CaCl and later on by electroporation incase salt based method wont bring any results. But I wanted to try out sonification since it is in a way easier and cheaper to build/acquire and perhaps safer)).I am also working on developing more energy efficient containment for the culture to use minimal energy for shaking and molecule extraction methods, so anyways I dont have a PhD or anything I am a undergrad student but you can probably use some fancy words if you feel like. I am interested if anyone ever used multiple techniques and has any feedback on it which ever it is bad or not.Thanks in advance.
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