Phage
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Genetic engineering and fast plants
So for my last biology course at the JC im attending ive been given the oppurtuninty to devise my own lab experiment. I have to use wisconsin fast
plants, which are increadibley fast growin little plants that mature in a matter of a few weeks.
My plan was to purchase Agrobacterium which had the Ti plasmid with the desired gene already in it (bio luminesence, roundup ready, any ideas?) and
then infecting sections of the leaf, growing the leaf out on plates, rooting and so on. there was also the possibility of infecting the flowers
directley in hopes that any seeds produced would carry the desired traits.
Problems! my teacher thinks its too complicated a project  but i personaly don
think so. the problems i have is that i dont know anything about raising the bacteria, i cant find any information on its use on wisconsin fast plants
and most importantly of all, i have yet to find a place that sells the bacteria with our with out the desired genes in them.
so if anyone has any ideas, im all ears. i looked into other methods and the most promising and equally if not more cool technique would be using a
Gene gun. however i have yet to find anywhere that sells them.
Any one ever think of making a gene gun? thier design is simple, the tuning may be a little hard.
thanks for the input everyone
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XxDaTxX
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Unless you have experience in the field, I doubt this experiment would be feasible for a lower division course in genetics (I am assuming that is what
this is for). If you want to ignore the advice given to you, wild-type Agro strain include A. radiobacter K84 as well as A. vitis S4. Either you or
your teacher would have to write to another college and ask for the strain. They are published, fully sequenced, and as such, you should not be
charged for it. It is somewhat odd asking for a wild type strain (most requests made to a lab are for strains in recent publications), so I would
suggest your teacher write to the appropriate lab at his/her alma mater. If you have no experience in microbiology, I would say you have a lot of work
cut out for you, to say the least. I'd help you if you were in my lab... I'd say save the enthusiam for the school you transfer to. Get into a
research lab early.
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Darkblade48
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Your project is feasible, provided that you find the correct strain (as XxDaTxX mentionned, asking a college/university should provide you with the
appropriate strain).
Using a gene gun would work, but probably would be (relatively) expensive to buy, and may be more of a hassle to construct. Transformation of the
plant cells simply by dipping cut leaves into a solution of the Agrobacterium strain is probably the easiest way.
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Phage
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so as far as the wild strains, what method would you recomend for introducing the plasmids with deactivated Ti sequences as well as the gene of choice
and a marker? im assuming there is somewhere i could buy the plasmid, then an ecoli production could be set up. from there whats the best method for
introducing the plasmid? simply mixing the two bacteria in a culture? i havnt looked into introducing the gene into the agrobacterium as of yet. if
anyone could point me in the direction of a book or web site about growing agrobacterium i would be ever so greatful. thanks for the advice!
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Darkblade48
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Assuming you have a strain of bacteria that has the Ti plasmid (recipient) and another strain that has the gene of interest (donor), you could
transform the recipient strain to introduce the gene of interest into the plasmid.
Alternatively, if you want to get more complicated, you could use transduction, but I assume the idea is to keep it (relatively) simple.
Growing the Agrobacterium shouldn't be too difficult; I'm not sure of the specific type of agar that is required for their growth, but if you check
the literature, it should say.
Be sure to also prepare a selective media (i.e. has Roundup in it), in order to select for the bugs that have the gene of interest (i.e. gene that
confers Roundup resistance, etc).
Edit: Transduction may or may not work; I'm not sure of the sizes of the genes you're trying to introduce, so be sure to keep that in mind as well
[Edited on 9-6-2007 by Darkblade48]
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Phage
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My teacher went ahead and took away my right to pick what im going to do for my project. She went ahead and bought Rosette-Dwarf (homozygous rececive
(ros/ros) for low gibberellin production) seeds. she then went on to tell me my project will consist of figuring out a method for treating the
dwarfism. ok so i treat them with GAA, increadibly lame, almost as lame as some of the projects other in my class have choosen such as seeing what
salt does to the plants growth. i told i her that id do it (i had to) but that besides simple GAA addition i would also investigate the process of
gene therapy for the plant. since this way i can not fail ( i can fall back on the GAA) She approved it.
so why am i telling all of you this? ive found this publication: http://www.freepatentsonline.com/20050245732.html i have yet to read through the whole thing but it looks promising. Im hoping now that i can
purchase the gene that encodes for gibberlin production and then go from there as if nothing had changed in my project design.
I also just started reading about Electroporation after treatment of the plant walls. i still have to do LOTS of reserch on this method since ive only
seen it done once and that was to E. coli. Is this a realitvley easy method compared to starting Agro cultures and so on? it seems like it would be.
last question. im brainstorming sources of the genetic material. im sure reading that whole article will help, how about removing segments from the
non dwarf plants that i have? ive done dna extraction before but as far as usable dna and only the sequences i need.... i could always purcahse the
nuclear material from a web site but i have yet to see this gene, let alone premade with a promoter and marker.
does anyone have experience with growing this plant? i starte da few seeds a few days ago. seems that the planting mix that the site sells is
actuallymuch better than peat moss (duh) but also much better than miracle grow sprouting mix!
thanks again.
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Darkblade48
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Electroporation is an efficient method of transformation, but will be expensive, since you need to purchase the equipment, and also have to have the
right conditions (too much electricity, or too long a period of electroporation will result in dead cells).
To be honest, I think the Agrobacterium method of transforming cells is fine; the other alternatives may be easier, but involve complicated equipment
that you may not have access to.
You could try to remove the segment that produces the gibberellin from WT (wildtype) plants; I would imagine this would involve using restriction
enzymes to properly digest the genome, followed by incorporation of the fragments into the Ti plasmid of Argobacterium. On selective media, you select
for the Argobacterium that contain the gene fragment of interest. Then transforming the mutant plant cells with the Argobacterium should result in
gibberellin production.
Of course, the entire process (which is quite lengthy) can be skipped if you could find a source for the gibberellin gene (I'm sure one exists; the
sequence should have been sequenced long ago).
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Phage
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yeah it looks like the gibberlin gene was definetly sequenced. i have several papers with it. now i jsut need a source. electroporation seems liek the
easiest transformation tek because a simple power supply is really all that is needed and im almost certain the school has one. plenty of plant
material will be at hand to experiment with so no problem there. of course the agrobacterium is a little more interesting so it gets a few points. hmm
now i have to decide. i think ill go with electroporation because that way i dont ahve to worry about culturing anything but the plants.
So my first step will be to start growing the plants so i have starting material for experimenting with (duh). Then i need to find a source for the
gene. the gene will ahve the gibberellin sequence on it with a promoter and im thinking an antibiotic marker. dumb question, how does the antibiotic
work on plants? i understand bacteria but why plants? is it because it attacks the choloplast which are related to bacteria because of their
endosymbiotic orgin? as you can tell im learning all of this stuff as i go and its only been about 3 days. next would be the power supply and
containers. a good plant growing medium needs to be worked out.
thank you darkblade48 for the help! you seem to knwo your stuff. what kind of projects are you working on in your lab?
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Darkblade48
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Electroporation is easy provided you have the correct equipment to perform it with 
Conversely, growing Agrobacterium is easy even without the presence of high tech equipment; the only disadvantage would bet hat transformation would
be less efficient, and time would be spent in growing/transforming the Agrobacterium first.
You may find out that the cuvettes for electroporation are quite pricey, but the choice of technique is up to you (or your funding source...)
I am not sure how antibiotics would work on a plant, as I've never actually done plant genetics; perhaps someone that has can comment. Your suggestion
certainly sounds plausible, but in the end, I am not sure.
Thanks for the comment, I would hope that I seem knowledgeable, afterall, I've been in the microbiology field for the last 3 years at university My current project in the lab involves studying Salmonella ser. typhimurium and its
modulation of host proteins.
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XxDaTxX
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Your linked marker would depend on if your method of transformation. I suggest you steer clear from tissue culture as it takes a few months for one
to be minimally proficient with it.
I really do think if you are truly interested, you should think about working with an academic lab that can provide you with better
resources/training. In the mean time concentrate on the genetics.
The most direct method, that is a sure fire proof-of-concept for "gene therapy" would be a simple complementation. You seem to somewhat have the
right idea, but your genetics is a bit shaky. First, "the gibberellin gene" as you have called it, implies that you speak of gibberellin
biosynthesis. Gibberellin biosynthesis is accomplished by a number of genes. On the other hand, the gene that you should be referring to is the ros
gene. This gene codes for a regulator for gibberellin production, rather than coding for biosynthesis as your word choice implies.
Generally, complementation using Agro would be by cloning in a wild type copy of ros into the T-DNA region. There is no "source for the gene" other
than the template obtained from a genomic prep from Agro cells. Amplify, digest, clone into Ti, infect (with several steps in between as you may find
out when you actually go to do it).
There are binary vectors that simplify lab protocols tremendously. You need to identify the right ones (host specificity, and appropriate markers)
then make the request if it is a published strain.
Antibiotics are used in culture. Again, I would not suggest trying to learn that in the short amount of time you have. You want what is known as "in
planta" transformations. Really though, I suggest in applying in a intersession/summer research program. You can get good hands on experience in
addition to a stipend many times.
[Edited on 7-9-2007 by XxDaTxX]
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Phage
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Yes, my understanding of molecular genetics is poor at best. I knew that the gene i was after is the ros gene, i wasnt sure of its exact purpose.
after reading into gibberlin it appeard to me that it wasnt "created" soley by that gene, so i really had no clue what its purpose was. Thank you very
much for clearing things up for me. the most exposure to genetics ive had is simple Mendel genetics. im just now teaching myself about how these genes
are controlled.
im not going to give up on this project, i dont know if im going to succed but im going to at least try. i ahve something to fall back on anyway, the
GAA is a sure fire way of "curing" the dwarfism. By source for the gene, im referring to a company that sells pre made plasmid dna contaning the
gene.
Patent 6794560 has some great info on the ros gene. i plan on reading the whole thing when i get home from work and after i finish the design for the
tesla coil me and friends are working on. XxDaTxX - you didnt mention anything about the electroporation, what do you think abou this method as
opposed to agrobacterium? my thoughts are that the less culturing i have to do (as you mentioned it takes time to learn tthe techniques for this) the
more of a chance i will be able to succed.
so before i do any more reading let me make sure i have this straight. and please understand that i realize im a complete noob to this field, im
pretty damn good at chemistry but biotechnology is brand spanking new for me. what i need to aquire is the ros gene and place it into the dwarfs
genome. since the ros gene codes for a regulator for gibberlin production it requires a promoter infront of it correct? sorry if i sound pretty damn
ignorant, this is all new to me. ill read a book or two on molecular genetics tonight, pinky swear!
what i really need to find now is a method for aquiring the ros gene alone. wether i can order it from an online store or somehow remove it from a
wild type plant of mine. ill start looking again.
sorry if i seem stuborn but my teacehr said it was to hard for me to accomplish, therefore i must 
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XxDaTxX
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Electroporation is a good method when working with cultures which, as I understand it, you have agreed not to bother with.
As for getting things straight, you are on the right track. Your ros-/ros- plant has two mutant copies of the Ros gene. You need to introduce one
working (wild-type) copy of the gene including its promoter. These things are not bought. You use PCR to amplify the upstream+coding sequence of ros
using DNA of your plant as a template. The only thing you order is the primers, and the enzymes for cloning it into your vector's T-DNA region.
Alternatively, you can go the traditional route (the one that the course is trying to teach you) and cross your dwarf with a normal plant (of unknown
genotype) and see the wild-type:dwarf ratio in the progeny to then determine the genotype of the normal plant (either ros+/ros- or ros+/ros+).
[Edited on 9-9-2007 by XxDaTxX]
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Phage
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thanks for the straigth forward answer. I have nothing against doing cultures. but having to do multiple cultures with different organisms is what im
trying to avoid. so electroporation is teh way to go . So now that youve repeated yoursefl several times im finaly seeing waht your getting at as far
as aquiring the gene. so now i will start reading up on the method you mentioned.
so im writing up my proposal right now. ill be growing out several dwarfs and several wild types. i will then cross these plants in order to see teh
genotypes of teh wild types. From the original dwarf plants i will attempt electroporation to introduce the working ros gene and its promoter in
segments of leafs wich will then be grown out on plates and so on. I will also grow dwarfs that are treated with GAA, so that then end product of the
gene therapy, The hormone tratment and the Wild type can all be compared.
thanks again for the help. im going to go write up my project proposal right now. ill keep everyoen updated, with pictures, on the progess of the
project (not sure if anyone cfares to see but im going to do it anyway  ). i
think it goes with out saying that there will be plenty of questinos to come.
thanks
chris
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XxDaTxX
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Actually I was trying to discourage the use of electroporation and cell culture. Look up in planta transformations. There are several protocols that
are based on wounding, infecting, then regenerating without ever having to culture.
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Phage
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haha, ok like i said it takes time for me to grasp what others are trying to tell me. i like what your saying. no PLANT cultures but instead do only
AGRO. cultures. alright ill give it a try. thanks
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Chalo
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preset for you, attached overview of plant biotech methods.
Attachment: agrobook 03 plant biotech.pdf (441kB)
This file has been downloaded 367 times
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