replacing Cells in and outs

[mehdi71000]

Hi everyone
Any good sources of luciferin expression vector?
and
I have this crazy idea. Is it possible to remove all the dna from a cell and insert another species dna and remove all cell parts and add new cell parts. Would the cell work? You might ask why. I thought it might be interesting. If so could I do the same but inserting a bacteria in a plant cell. Thought this might not be possible since cell walls have protein in them witch transfer food to the cell and plant cells might need different nutrients.
Thanks for your help

[schizophrenicsinner]

Don’t become all Dr. Jekell & Mr. Hyde !

DNA is not to be tampered with: I don’t know what your teachers tell you, but don’t!

A doctor’s vow is to heal: For this you fix & let heal… Don’t tamper with nature.

Schizo8

[MrMistery]

cloning is something like this: take out the nucleus from an egg cell and put in a diploid one from a somatic cell. However, it must be from a cell of the same species. Putting a bacteria in a plant cell will lead to immediate cell death, no doubt about that.

[mehdi71000]

thanks guys
is there an expeiment i can do in microinjection that does not require vector injection. i need this becouse the vector i orderd is going to take few weeks to arive.
thanks

[satrohraj]

quote mehdi71000:

Hi everyone
I have this crazy idea. Is it possible to remove all the dna from a cell and insert another species dna and remove all cell parts and add new cell parts. Would the cell work? You might ask why. I thought it might be interesting. If so could I do the same but inserting a bacteria in a plant cell. Thought this might not be possible since cell walls have protein in them witch transfer food to the cell and plant cells might need different nutrients.
Thanks for your help

It will surely lead to death if the DNA is not compatible, which is the most possible case!

[mehdi71000]

What if a cell dna and other parts are removed and your only left with the nucleus wall and the cell wall. Why would the cell die since its all molecules? Does the cell not function at all? Is that called a dead cell? What if after cells death some one injected the same spices dna and cell materials. Would the cell function again?

[blcr11]

There were some classic experiments done–I forget exactly when, possibly as early as the early 1900s–involving the transfer of the nucleus of one cell type into a de-nucleated cytoplasm of another cell either of the same or different type. My (now dim) recollection of the experiments is that they were trying to see which was dominant in development, the nucleus or the cytoplasm. They may also have looked at or for cytoplasmic factors affecting the cell cycle–can’t say for sure anymore. The point is that similar experiments have been done–though not between plant and animal cells that I recall. My guess is you could get it to work maybe between different types of animal cells, though how distant a relationship could be tolerated I couldn’t say. I’m skeptical it would work between plant and animal cells. It’s also not immediately clear to me what you could do with such hybrid cells, but I’m willing to be convinced. Re using "dead" cells–there I doubt anything would work. Well, how dead? Nearly dead or mosty dead? If you transfer a functional nucleus into a non-functional cytoplasm, how can there be any translation? And conversely, if you put a dead nucleus into a fully functional cytoplasm, where will the mRNA come from to instruct the protein synthetic machinery?

[mehdi71000]

I thought cells and protein work as a result of chemical reactions. So I assumed a dead cell is a cell that its chemical reactions are distorted some how and it cant function. if this is true if I remove the nucleus content and the outer nucleus content the walls will still be functional but with out a energy source would be dysfunctional.
I all so though if a cell dna is changed if it duplicates would the new wall be the wall of the introduced dna. I mean the dna is responsible for the building of the cell wall so with the new materials how can the cell wall split to form 2 cells? I think I didn’t thought to much about this topic before I asked a bout it. I assume every living thing cell walls function differently when the cell splits. is this true?
Any cheap sources of lusfres vector those that glow ?

Thanks for your replies

[david23]

how do I answer this one

[blcr11]

An organism dies and, for some period of time, individual cells may remain functional. That’s why it is possible to transplant an organ from a “fresh” cadaver. When an individual cell dies—whether it dies “naturally” or due to external damage—it has lost essential functions: its chromosomes have lost the ability to pair or divide, or the membranes are compromised such that metabolism is not possible, or—who knows exactly why the cell is no longer viable. Some enzymes or structural proteins may remain more or less intact (but probably not for long since one of the things that happens pretty quickly is lysosomal disintegration and release of lytic enzymes into the surroundings accelerating the loss of functional proteins) but the logic is missing. It’s hard for me to imagine restoring enough function by the experiment you’re proposing. I wouldn’t categorically deny the possibility, but I think you’d have a hard time convincing NIH or NSF to fund you.

Invitrogen, Clontech, Promega, possibly Roche Diagostics sell luciferase related things—but I doubt any of them will be all that cheap.

[mehdi71000]

Thanks man you’re a life saver
in these two sites witch is best for making glowing plants and glowing animals by luciferase vector?

Site A:

http://www.promega.com/search/Default.a … +vector%22

Site B:

http://www.promega.com/catalog/catalogp … cer+Vector

site C:

http://www.promega.com/catalog/catalogp … say+System

i think site B is suitable. But I’m not sure. I’ve read and they said I think its for mammalian animals 😕 Could I use it on plants in cell culture and bacterias aswell
Thanks guys

pGL2-Enhancer Vector

http://www.promega.com/tbs/tm003/tm003.html

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[blcr11]

Try this: http://www.promega.com/pnotes/44/luerhsen/luerhsen.html

My guess is you can’t use a mammalian cell-specific promoter in a plant cell, but what do I know about plants?

[mehdi71000]

Thank mate
I read the PDF and I found the products below in it. Witch one do you think is the glow vector?
Thanks

http://www.promega.com/catalog/catalogp … say+System

[mehdi71000]

If I’m not mistaken
Luciferase Cell Culture Lysis 5X Reagent
Is suitable for plants and every thing else.

"The Luciferase Assay System may also be used for quantitation in plant and bacterial cells, but only Cell Culture Lysis Reagent is suitable for these applications"

Or is it best I should purchase Luciferase Assay System 100 assays. Witch I think has every cell in it. 😕

I’ll gives them a call tomorrow and keep you posted if you’re interested.
Thanks you all for your helpful replies

[mehdi71000]

ok this is the reply i ve got:
Thanks for contacting Promega technical services. I think that you may be a little confused about what you are after here. We have firefly luciferase vectors under the control of the strong viral SV40 promoter which would be able to express the luciferase gene in plants. However, they will not “glow” unless the substrate for luciferase is present as well. The substrate is luciferin and this is not expressed in plants so just expressing luciferase will not lead to any bioluminescence. The other thing to consider is that even if the luciferin is present the plants will not glow as the half life of the reaction is very short (2-5 minutes). Could I ask why you want such a vector and why you want to produce plants that will “glow in the dark”? I think you may be looking for a Green Fluorescent Protein (GFP) expressing vector. This vector have been used in the past to label cells and organisms and will produce a green fluorescence when excitation and emission are provided by the correct UV source.

😕

But I’m sure they have made glowing tobacco plants . Haven’t they?

[david23]

hahhaha, see how important it is to have GFP. Ask if they have cheap GFP. The only difference I see from the reply is that GFP glow lasts longer. Also ask if they have YFP and CFP as well. If you already bought the luciferase, keep it. You can use it for other stuff.

[mehdi71000]

What if I add luciferase expression vector + luciferin expression vector. So the luciferin is available for reaction. And since the cell keeps growing and making now enzymes and proteins the reaction will last? You think would it work?
They said the luciferase will work in mammalians but they said they’ll get back to me on that.
I mean what’s the point of a fish that glows that it doesn’t glow in the dark. only under uv.
what can i use luciferase for?

[david23]

forget the UV thing. The half life thing is more of a concern but who cares about that. luciferin is added separately. No need for vector. Luciferin is not sold in vectors, it’s not even a gene.

http://en.wikipedia.org/wiki/Luciferin

Look at the picture, it’s not an amino acid, it’s made separately by other mechanisms. To satisfy your glowing plants you would have to constantly add more and more luciferin each day. GFP dont have this problem. Save the luciferase for other experiments that you can play with later, if you want to do genetic engineering for real with those plants or your fish.

[mehdi71000]

I see your point but there must be an enzyme or a protein that create this chemical. Any ideas what it might be?

[david23]

There are, but it’s most likely a lot of enzymes and too many substrates involved. You are making this more difficult.

[mehdi71000]

No worries I’ll email some companies and ask them if they have such vector. it would be much easier to know the name of the enzymes responsible maybe they have a vector fully developed that caries several genes for all the enzymes witch produce luciferin, witch in results makes luciferin in cells

[david23]

it’s not that simple and not premade.

[mehdi71000]

Ok I asked the company and they said that they don’t have the luciferin substrates expression vector. So I guess ill keep on looking

Ok this may sound silly and promise you don’t laugh, I don’t know much about DNA.
I read some ware last year so I’m not sure if I’m right. It said genes start 20 something letters before ATATA or something same. If this is true could one break the dna at points. In result every broken peace has 1 gene in it. and by adding all of these broken dna into a cell. What are the chances of the cell expressing the ones that it can? For instance. we are looking for a gene A but we have as a result of splitting the dna we have genes A B C D and F. so buy placing all of them into a Cell if the gene is for an enzyme witch can be expressed, that one will be expressed. And the some others will be expressed as well but the desired gene A did get expressed?so in result I may end up with a cell with the protein A but also with a muscle protean .I can feel I’m of course a but would it works?

[mehdi71000]

http://www.freepatentsonline.com/8270.html

[david23]

breaking up like that isnt that likely, but there are could be other problems, which is why I asked you to clone a lot of it first, and when you inject try to do it with multiple cells.

Are you still obsessed with luciferase, it’s not superior to GFP in glowing plants and animals. You are getting into too much trouble.

[mehdi71000]

Have your heard about Aequorin
From what I found. it requires only calcium to glow. I don’t think plants have that much calcium in them but what do I know.

[x__Dmitri]

What’s this about calcium? I drink 4 glass of milk a day. I’m good, my bone mass isn’t going to disintegrate. And for all you soy milk drinkers… go suck a cow. Cow milk is real milk!

[mehdi71000]

Ok
There is an enzyme called luciferin-regenerating enzyme (LRE) this enzyme with the aid of D-cysteine regenerates luciferinoxide into Lucifer in. this reaction is common they said in fire fly.
what is D-cysteine is it an amino acid?
Any way david23 your right it’s too bloody complex here is the link
http://www.jbc.org/cgi/content/full/276/39/36508

Any ideas on Aequorin. From what I heard its not half bad

[david23]

D cysteine is an amino acid, could try. The diagram on that article uses multiple enzymes, more genes for you to use.

[mehdi71000]

thanks for your help guys wish I could repay you some how 😳

[mehdi71000]

Have your heard about Aequorin?

[david23]

i have no idea, did u spell it right

[mehdi71000]

Yes it’s from a jelly fish called Aequorea victoria. It needs calcium to emit light .would it work with plants if they are fed calcium? I found some calcium containing fertilisers as well
If you like I can buy some and clone some for you swell.
Scroll down to the second segment Aequorin-based Assays

http://las.perkinelmer.com/Catalog/defa … lAodb1LVVw

http://images.google.co.uk/images?hl=en … a=N&tab=wi

http://en.wikipedia.org/wiki/Aequorin

GUS CAT NEO genes they glow as well. But no clue were to buy them from

[david23]

good idea, try that if u can

[mehdi71000]

Can you direct me witch one the products is the actual vector. please
Thanks

http://las.perkinelmer.com/Catalog/defa … lAodb1LVVw

[mehdi71000]

I found some GM trees in the internet. They are supposed to be super fast growing type. Is the gene responsible in a separate vector or you think it’s very complex and inserted into the actual DNA. I ask this because if I could extract the responsible vector and create my own fast growing plant would be interesting.

[david23]

u cant extract any vector, u extract the genes and then put into a vector to put into another plant. You will need some extra expensive equipment and enzymes to put genes into vectors.

[mehdi71000]

Thanks pal your the best
I checked there plant there are not actually GM they are cross pollinated.

[david23]

if u can cross pollinate that plant with your glowing plant later it might be great.

[mehdi71000]

nice idea

quote mehdi71000:

Can you direct me witch one the products is the actual vector. please
Thanks

http://las.perkinelmer.com/Catalog/defa … lAodb1LVVw

any ideas on the link above?

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