Electroporation vs Sonification vs CaCl Chlorella Vulgrais

[Alex D]

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. 

[Alex D]

test

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[Sebastian S Cocioba]

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

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[Alex D]

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. 

[Sebastian S Cocioba]

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!

Chlamy.org/methods.html

Or just Chlamy.org

Sebastian S. Cocioba

CEO & Founder

New York Botanics, LLC

Blog: ATinyGreenCell.com

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[Alex D]

haha no prob, there is a paper ''acrobacterium mediated transformation of chlorella'' but i didnt get to it yet, so there has to be a way. I probably wouldnt stop just at getting the plasmid inside and i was thinking later on to incert agro's 2 proteins into the plasmid simultaneously with another CMV promoter so once plasmid gets inside and reports with luciferin that it is inside to start producing the two proteins that agro has which cover the plasmid and the other one that takes it into a nucleous. Pretty much like a virus with intergrase but ''integrase'' will be make inside before the integration of the dna. I also was thinking to encode the whole cas9 to be made inside post transfection but that is probably next year or something. 

I was told pretty much by everyone that i should do chlamidomonas but is has sexual reproduction so it got me scared that integrated gene might be lost. Vulgaris on the other hand is super stable from what i could see it but it is missing the flagella, it does have genes for flagella and sexual reproduction but for some reason they are silenced, maybe it is because it lives inside the paramecium... not sure. As far as my own  test culture it has to be shaken once an hour or so to homogenize it a bit otherwise it barely grows in static with purple and red light. my second culture i had was under room conditions mixing it once an hour or so but i didnt have much medium left and i kind of for the sake of experiment pored a coconut water into the tube and the thing started to grow like crazy... obviously i have found out later that it has been done before and some people use coconut water for plant tissue culture but it is fairly recent discovery maybe 10-15 years or so. But one Liter cost me $1. 

Another thing about chlorellas alot of them are resistant to major antibiotics and i have no idea how did it happen though out its development, the only one i have found so far people use it chloramphenicol , but overall chlorellas very similar to yeast its euk, has chitin like cell walls but the most what made me fall inlove that its chloroplast is some say most efficient thoughtout the whole plant kingdom. So thats probably why i didnt give up on it lol. 

OK this is gonna be creepy but few months back i made a presentation about use of algae for medicinal molecules in NJ and we actually talked about the open insulin project too. I am a biotech student so lol 

[poli]

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)?

[Alex D]

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 lol

From 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? 

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[John Griessen]

On 12/17/2015 12:00 PM, Alex D wrote:
> 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

Longer as in minutes is probably just going to cook/kill most anything.
2kV is the high end of the range for non-salty suspensions, so safety is the hurdle.

I'm working on ways to get low volumes of plastic parts "molded by John" and that will lead
to some lab gear with safety interlocks in a year maybe.

You could try using a barbeque grill piezo spark lighter and various R and C to get different volt levels
to your cuvette, but you sound impatient and "try anything", so I have to say "Please don't -- so you don't
forget and grab the thing being electrocuted with one hand and the spark hot wire with the other and
give yourself an arm to arm shot that puts your heart in fibrillation leading to death."

Instead of getting in big trouble, buy the Biorad Gene pulser here: $175 http://www.ebay.com/itm/like/221303599697

$39 (needs work) http://www.ebay.com/itm/Bio-Rad-Gene-Pulser-Laboratory-High-Voltage-Electrophoresis-Unit-1652076-/361451197470

$85 untested http://www.ebay.com/itm/BIO-RAD-GENE-PULSER-/262178386007?hash=item3d0b0cb057

$139 extra huge cap bank: http://www.ebay.com/itm/Bio-Rad-Capacitance-Extender-1652087-Gene-Pulser-Electrophoresis/201094768798

Be sure to read the destructions even with the safety engineered Biorad machines. Those cables between can be deadly.

[Alex D]

Hello John, 

well the power supply for the electrophoretic unit we have during gel pulls about 85 volts and i believe 0.8 Amps if im not mistaken. Theoretically if buffer is present and just by placing a 50cent cuvette  into the electrophoretic chamber it should work. We do have functional electroporator from  BTX but i was trying to figure out the way to avoid using it due to its price. 

http://www.coleparmer.com/Product/BTX_Electroporation_System_Square_Wave_100_240_VAC/UX-02894-18?referred_id=778&gclid=CL2Bm-zR5ckCFdUSHwodngIEqQ

that is the unit we have. But i am leaning more towards the sonication method since its fairly easier to replicate. I am more interested why do people choose eletroporation or sonication.

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[John Griessen]

On 12/18/2015 08:50 AM, Alex D wrote:
> Theoretically if buffer is present and just by placing a 50cent cuvette into the electrophoretic chamber it should work.

Have you analyzed that with the resistivity concept? Where longer paths made of tiles of the same resistivity square
of material have higher and higher resistance? Sounds like a possible way to get zapped while fiddling with unknowns and
getting in a hurried mood.

We do
> have functional electroporator from BTX but i was trying to figure out the way to avoid using it due to its price.

Is its price in the range of the super cheap < $200 ebay prices for Biorad "classical" electroporators?
If so, that's cheap enough.
Going lower is OT for this list, isn't it?

[Alex D]

I am not pursuing of ''zapping myself'' so i think those calculations are irrelevant, besides that I never seen anyone touching cuvette with two hands  and if it will be touched with one the current will go straight from one finger to another and back to the cathode. And while I have access to brand new $8000 piece of equipment I dont really see a point of getting some molded rusty box of a cat in a sack. Besides i can build sonicator myself for under $50 so i am deffinately not spending hundreds on electroporator. 

[Mega [Andreas Stuermer]]

You said you're going to use CMV promoter? Better don't, it seems to get confused with CaMV promotor (35S) from time to time. The former one is from the latent Cytomegalovirus which infects mammals and works in vertebrates and insects, and the other one from the Cauliflower mosaic virus which is active in plants.

Wouldn't it be easier to start with pCambia1302? It already contains all the elements you need.

[John Griessen]

On 12/18/2015 11:25 PM, Alex D wrote:
> I am not pursuing of ''zapping myself'' so i think those calculations are irrelevant,

No one ever pursues unsafe conditions and I never suggested you would, just that casting about for quick
ways to get to point B is not a good safety recipe so you should stick with electroporator products instead
of building it yourself.

besides that I never seen anyone touching
> cuvette with two hands

It's not the cuvette, its the wires between modules. You were suggesting to use an electrophoresis supply and gel box it seemed
to me...

If you understand the one handed approach to touching anything high voltage and have that really at
a gut level you'll probably be safe.

I didn't see that anyone has responded to the original question yet.

Is sonication just as good as electroporation?
Probably depends on the recipe. Electroporation can be done in a flash is one of its pluses.

[Alex D]

Yea i have noticed that actually, and yea we can use many common plasmids but we probably will just order custom to avoid restriction use as much as possible. Recent study showed that chlorellas chromosomes can replicated in mammalian cells too and use of the promoter doesnt induce any infections or posses threat. SV40 promoter was used for many years in gm fish many years back   

[Alex D]

OK for the 4th time I AM NOT BUILDING THE ELECTROPORATOR....

[Alex D]

If you have used elecrophoretic units especially latest ones, it is nearly impossible to get electrocuted, all chambers must be closed fully, once you pull the cables they automatically locked by the enclosure.    

On Saturday, December 19, 2015 at 7:19:14 AM UTC-8, John Griessen wrote:

[poli]

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. 

[John Griessen]

On 12/19/2015 10:51 AM, poli wrote:
> The nanno oceanica I work with

Is your suspension for electroporation salty and conductive? What kind of voltage and waveshape and number of repetitions
do you use?

I'm interested for purposes of designing low-cost-super-safe electroporation equipment. So far, the common uses
are for non-salty suspensions so that very low power pulses work. If salty and conductive, most of the energy goes into
warming up the solution and pulls volts down unless you supply much more energy to the suspension.

[Alex D]

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.  

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[poli]

The algae is washed and resuspended in 375 mM sorbitol. The biorad gene pulser ii electroporation settings we use for a 2 mm cuvette are 2.2 kv, 600 ohms, and 50 uf, and I think the pulse is an exponential decay. We use a single pulse and get good transformations with time constants from 20-30 ms.

[Zulfiquer Jahangir]

Using hygromycin B resistance as a marker for selection establishes the conditions required for the transformation of Chlorella vulgaris. Some scientists  exponentially grown C. vulgaris cells were transformed by electroporation with plasmid pIG121-Hm, and transformants were selected with hygromycin B at a concentration of 50 μg/ml. Cell extracts prepared from the late-log cultures of the transformants exhibited glucuronidase activities as conferred by the gus gene on pIG121-Hm. The maintenance of plasmid in the algal cells seemed to be transient as many cultures derived from the hygromycin B-resistant colonies gradually lost the hygromycin resistance upon prolonged growth. The result of Southern blotting of the genomic DNAs prepared from transformant cultures exhibiting persistent hygromycin resistance showed that integration of part of the plasmid DNA into the host chromosome had taken place.

On Wednesday, December 16, 2015 at 1:46:09 PM UTC-5, Alex D wrote: