yep we will be using the same plasmid for both, two promters are cmv and NR. But i am also very curious to test many other promoters as well as mammalian but im not sure if it is possible to put restriction cites on the promoter ends or I will need to replace the promoter together with the gene. The reason i wanted to try just salt is because ''theoretically'' electroporation and sonication doesnt destroy the cell wall on low level so maybe it is possible to pore the membrane and get plasmid in i know it is a long shot but it wont cost much to try couple times.
I also thought of making a linear plasmid that it might increase the penetration by cutting it and then make loops at its ends similar to viroid or satellite shape and i also had an idea of adding the proteins from Ti to somehow perhaps keep it linear but that is more like next step. I have seen a paper and i have it that someone did transfection with use of agrobacterium but i didnt get to it yet but i think hybrid plasmids or plasmid/protein might increase the penetration or assist it in someway. I kinda want to learn the protocol first get used to it and get the initial data on efficiency etc. and then start to experiment with multiple promoters, perhaps 3+ on same plasmid maybe but it might increase the size beyong 10k.
But nanno is probably will be a cheaper specie for protoplast preporation? as far as i have found it has cellulose lignin and alganan ( which is extremely interesting to me at this point ) I might have extra funds to get the enzyme and try it but it will be sometime next summer.
funny thing is , i just ran two cultures of C vulgaris and nannochloropsis ( not sure what specie ) i got it from corolina , after leaving chlorella it died with in two weeks or so dark yellow brownish goo.. but nannochloropsis after a hand shake came back into solution all nice and green , i didnt do any cell counting just looked under a microscope it seemed pretty alive to me. Even though vulgaris supposedly has to have a michonism of ''hybernation'' during winter months in soil.
On Sat, Dec 19, 2015 at 8:51 AM, poli <polinere@gmail.com> wrote:
My experience has been with Nannochloropsis, I transform with electroporation but tested sonication a bit. Electroporation seems more standardized than sonication but both work to some degree. Electroporation seems to be more efficient but sonication is a simpler set-up (no need to do sorbitol washes). If you have a good transformation construct and access to the equipment testing transformation methods seems feasible. I kind of doubt chemical transformation will work, at least without preparing protoplasts which would can be quite tricky (time consuming, and the enzymes used are going to be expensive).--It seems like the first step for your project is to get a plasmid that will express in your organisms, chlorella and nanno are quite different evolutionarily (green vs heterokont) so they may not like the same promoters. I have always wanted to know what viral promoters work in nanno, so if you find out please update us. The nanno oceanica I work with seems pretty resistant to chloramphenicol, we use hygromycin and zeocin. Zeocin is a near universal selection agent (toxic) and would probably work with chlorella too. I am not sure if you were trying to one plasmid for both species or not, maybe you can give us a better idea of desired plasmid characteristics.To get DNA inserted in the nuclear genome we transform with linear DNA, to get it to go to the chloroplast you would need to add homology arms. Chlorella looks like it will integrate circular DNA but linearizing first may increase transformation rates.
On Thursday, December 17, 2015 at 1:00:24 PM UTC-5, Alex D wrote:hello,we dont have the pladmid yet to say exact size but we are putting CAT ( chloramphenicol Acetyl Trans) CMV promoter, euk and proc ORI and lucifirase I believe it should be somewhere a pinch under 6k. We only have the BTX electroporator in the lab and the protocol is pretty much straight forward even for chlorella vulgaris from what I could find from papers but the cheapest new units are starting from around 2k for the eppendorf. The reason i wanted to experiment because from my ''unaducated guess'' electroporation only produces cavitation in the plasma membrane and leaves matrix and outer cell walls untouched ( i assume) so I pressumed that CaCl could be used aswell at least worth the shot to make the process way cheaper.As far as killing chlorella its very simple by sonication many places sell sonicators against the algae blooms but they are too strong and they break everything including the cell membrane but much cheaper then any eletroporator, so i was thinking if I acquire less strong transducer it will allow me to disrupt the membrane enough to create pores. This way price of the protocol would be nearly as price for salt method but faster and more efficient probably.About the capacitance, resistance, and field strength, I am not sure as far as the info i have found for now just indicates i believe volts per cubic inch or mm or amps per cubic mm not sure but I have seen many papers have some sort of formula helping to determine the time of exposure depending on the voltage. As far as my mentor told me he was using that BTX with 2.5KV for 50 mili sec, but i have seen papers where people have used 150 200 volts but i think for the longer time or smaller cuvette? I proposed to give it a shot for a minute or two in inside the gell in the electrophoretic unit and then check the results but that kind of went against the schools lab safety policy lolFrom what I have researched so far it seems that except the salt the sonicator would be the cheapest and safest method.. if it works lol so i am deff. will try to build one this winter. the electroporator i am just keeping as my plan B because I have to actually make a presentation and deliver results later on.The ''resistance cassettes, expression cassettes'' and the epitope i dont know what that is, does it have anything to do with the IGGs? for the blotting or something?On Thu, Dec 17, 2015 at 8:26 AM, poli <poli...@gmail.com> wrote:Electroporation works well for Nanno and is very straight-forward if you have a biorad gene pulser or similar electroporator. Do you have an electroporator that lets you change capacitance, resistance, and field strength? From what I have heard Chlorella is extremely difficult to transform but I don't have personal experience with it. Do you have the transformation vectors, if so can you describe them (resistance cassettes, expression cassettes - promoter, epitope, terminator)?--
On Wednesday, December 16, 2015 at 1:46:09 PM UTC-5, Alex D 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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