Re: [DIYbio] Electroporation vs Sonification vs CaCl Chlorella Vulgrais

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. 

Sebastian S. Cocioba

CEO & Founder

New York Botanics, LLC

Blog: ATinyGreenCell.com

On Dec 16, 2015, at 1:49 PM, Alex D <alexx...@gmail.com> wrote:

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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