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Author: Subject: Genetic engineering and fast plants
Phage
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[*] posted on 4-9-2007 at 18:15
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 :mad: 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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[*] posted on 4-9-2007 at 21:54


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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[*] posted on 4-9-2007 at 23:55


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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[*] posted on 5-9-2007 at 21:43


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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[*] posted on 5-9-2007 at 23:58


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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[*] posted on 6-9-2007 at 18:14


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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[*] posted on 6-9-2007 at 20:06


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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[*] posted on 6-9-2007 at 21:15


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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[*] posted on 7-9-2007 at 03:24


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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[*] posted on 7-9-2007 at 22:45


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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[*] posted on 8-9-2007 at 11:05


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 :D
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XxDaTxX
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[*] posted on 9-9-2007 at 11:07


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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[*] posted on 9-9-2007 at 15:22


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 :P ). i think it goes with out saying that there will be plenty of questinos to come.

thanks

chris
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[*] posted on 9-9-2007 at 16:09


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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[*] posted on 9-9-2007 at 18:46


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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[*] posted on 27-8-2022 at 18:33


preset for you, attached overview of plant biotech methods.

Attachment: agrobook 03 plant biotech.pdf (441kB)
This file has been downloaded 41 times

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